Rheumatoid arthritis extra-articular lung disease: new insights on pathogenesis and experimental drugs.

INTRODUCTION: Pulmonary involvement is one of the most common extra-articular manifestations of rheumatoid arthritis (RA), a systemic inflammatory disease characterized by joint swelling and tenderness. All lung compartments can be interested in the course of RA, including parenchyma, airways, and, more rarely, pleura and vasculature. AREAS COVERED: The aim of this paper is to review the main RA lung manifestations, focusing on pathogenesis, clinical and therapeutic issues of RA-related interstitial lung disease (ILD). Despite an increasing number of studies in the last years, pathogenesis of RA-ILD remains largely debated and the treatment of RA patients with lung involvement is still challenging in these patients. EXPERT OPINION: Management of RA-ILD is largely based on expert-opinion. Due to the broad clinical manifestations, including both joints and pulmonary involvement, multidisciplinary discussion, including rheumatologist and pulmonologist, is essential, not only for diagnosis, but also to evaluate the best therapeutic approach and follow-up. In fact, the coexistence of different lung manifestations may influence the treatment response and safety. The identification of biomarkers and risk-factors for an early identification of RA patients at risk of developing ILD remains a need that still needs to be fulfilled, and that will require further investigation in the next years.

Development and Validation of Novel Z-360-Based Macromolecules for the Active Targeting of CCK2-R.

The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives (IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound (IP-002H-Rho) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R+) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002x-Rho association with A431-CCK2-R+ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002G-Rho and IP-002L-Rho, respectively. Unexpectedly, IP-002H-Rho showed a similar cell association to that of IP-002L-Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R+ cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho, respectively, proving IP-002M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R+ cells incubated with IP-002M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002M-Rho by A431-CCK2-R+ cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.

Single-cell transcriptomic analysis to identify endomembrane regulation of metalloproteins and motor proteins in autoimmunity.

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.

Identification of two autoantigens recognised by circulating autoantibodies as potential biomarkers for diagnosing giant cell arteritis.

OBJECTIVES: Giant cell arteritis (GCA) is a common vasculitis affecting patients aged 50 and older. GCA leads to chronic inflammation of large/medium-sized vessel walls with complications such as permanent vision loss and risk of stroke and aortic aneurysms. Early diagnosis is crucial and relies on temporal artery biopsy (TAB) and ultrasound imaging of temporal and axillary arteries. However, these methods have limitations. Serum biomarkers as autoantibodies have been reported but with inconclusive data for their use in the clinical setting. Additionally, C-reactive protein and erythrocyte sedimentation rate are non-specific and limited in reflecting disease activity, particularly in patients treated with IL-6 inhibitors. This study aimed to identify serum autoantibodies as new diagnostic biomarkers for GCA using a human protein array. METHODS: One commercial and one proprietary human protein array were used for antibody profiling of sera from patients with GCA (n=55), Takayasu (TAK n=7), and Healthy Controls (HC n=28). The identified candidate autoantigens were purified and tested for specific autoantibodies by ELISA. RESULTS: Antibodies against two proteins, VSIG10L (V-Set and Immunoglobulin Domain Containing 10 Like) and DCBLD1 (discoidin), were identified and found to be associated with GCA, with an overall prevalence of 43-57%, respectively, and high specificity as individual antibodies. A control series of TAK sera tested negative. CONCLUSIONS: Detecting GCA-specific autoantibodies may offer a new, non-invasive tool for improving our diagnostic power in GCA. Even though cell-mediated immune responses are crucial for GCA pathogenesis, this finding opens the way for investigating the additional role of humoral immune responses in the disease.

Elevated NET, Calprotectin, and Neopterin Levels Discriminate between Disease Activity in COVID-19, as Evidenced by Need for Hospitalization among Patients in Northern Italy.

Coronavirus disease 2019 (COVID-19) displays clinical heterogeneity, but little information is available for patients with mild or very early disease. We aimed to characterize biomarkers that are useful for discriminating the hospitalization risk in a COVID-19 cohort from Northern Italy during the first pandemic wave. We enrolled and followed for four weeks 76 symptomatic SARS-CoV-2 positive patients and age/sex-matched healthy controls. Patients with mild disease were discharged (n.42), and the remaining patients were hospitalized (n.34). Blood was collected before any anti-inflammatory/immunosuppressive therapy and assessed for soluble C5b-9/C5a, H3-neutrophil extracellular traps (NETs), calprotectin, and DNase plasma levels via ELISA and a panel of proinflammatory cytokines via ELLA. Calprotectin and NET levels discriminate between hospitalized and non-hospitalized patients, while DNase negatively correlates with NET levels; there are positive correlations between calprotectin and both NET and neopterin levels. Neopterin levels increase in patients at the beginning of the disease and do so more in hospitalized than non-hospitalized patients. C5a and sC5b-9, and other acute phase proteins, correlate with neopterin, calprotectin, and DNase. Both NET and neopterin levels negatively correlate with platelet count. We show that calprotectin, NETs, and neopterin are important proinflammatory parameters potentially useful for discriminating between COVID-19 patients at risk of hospitalization.

miR-146a and miR-146b regulate the expression of ICAM-1 in giant cell arteritis.

Giant cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries. MiRNAs are small, non-coding RNAs that inhibit gene expression at post-transcriptional level. Several miRNAs have been shown to be dysregulated in temporal artery biopsies (TABs) from GCA patients, but their role is unknown. The aims of the present work were: to gain insight into the link between inflammation and miRNA up-regulation in GCA; to identify the role of miR-146a and miR-146b. Primary cultures from TABs were treated with IL-1ß, IL-6, soluble IL-6R (sIL6R), IL-17, IL-22, IFNγ, LPS and PolyIC. Correlations between cytokine mRNA and miRNA levels were determined in inflamed TABs. Primary cultures from TABs, human aortic endothelial and smooth muscle cells and ex-vivo TAB sections were transfected with synthetic miR-146a and miR-146b to mimic miRNA activities. Cell viability, target gene expression, cytokine levels in culture supernatants were assayed. Treatment of primary cultures from TABs with IL-1ß and IL-17 increased miR-146a expression while IL-1ß, IL-6+sIL6R and IFNγ increased miR-146b expression. IFNγ and IL-1ß mRNA levels correlated with miR-146a/b levels. Following transfection, cell viability decreased only in primary cultures from TABs. Moreover, transfection of miR-146a/b mimics increased ICAM-1 gene expression and production of the soluble form of ICAM-1 by primary cultures from TABs and by ex-vivo TABs. ICAM-1 expression was higher in inflamed than normal TABs and ICAM-1 levels correlated with miR-146a/b levels. Expression of miR-146a and miR-146b in GCA appeared to be driven by inflammatory cytokines (e.g. IL-1ß, IFNγ). miR-146a and miR-146b seem responsible for the increase of soluble ICAM-1.

Eosinophilic giant cell arteritis: A different subset of disease?

OBJECTIVES: To describe the clinical findings, response to therapy and course of patients with transmural eosinophilic infiltration at temporal artery biopsy (TAB). METHODS: The study consisted of a retrospective cohort of 254 consecutive GCA patients with evidence of transmural inflammation at TAB seen at the Santa Maria Nuova Hospital over a 28-year period. The findings of the 22 patients with eosinophilic infiltration (≥ 20 eosinophils/hpf) at TAB were compared with those of 232 patients without. Among these 232 patients, we sampled 42 GCA patients matched for age, sex and follow-up duration to the 22 with eosinophilic infiltration, to compare allergic manifestations. RESULTS: GCA patients with eosinophilic infiltration compared to those without presented more frequently cranial symptoms (p = 0.052), headaches (p = 0.005), abnormalities of TAs at physical examination (p = 0.045), jaw claudication (p = 0.024), and systemic manifestations (p = 0.016) and had higher CRP levels at diagnosis (p = 0.001). Regarding histological lesions, a severe transmural inflammation, laminar necrosis and intraluminal acute thrombosis were more frequently observed in patients with eosinophilic infiltration (p = 0.066, p < 0.001, and p = 0.010, respectively). Long-term remission and flares were similar in the two groups. When 21 GCA patients with eosinophilic infiltration were compared to 42 without, blood eosinophilic counts at diagnosis were normal and no patients had evidence or developed allergic manifestations and/or clinical findings of systemic necrotizing vasculitis. CONCLUSION: Patients with transmural eosinophilic infiltration represent a subset of GCA with cranial disease and more severe inflammation.

CCL18, CHI3L1, ANG2, IL-6 systemic levels are associated with the extent of lung damage and radiomic features in SARS-CoV-2 infection.

OBJECTIVE AND DESIGN: We aimed to identify cytokines whose concentrations are related to lung damage, radiomic features, and clinical outcomes in COVID-19 patients. MATERIAL OR SUBJECTS: Two hundred twenty-six patients with SARS-CoV-2 infection and chest computed tomography (CT) images were enrolled. METHODS: CCL18, CHI3L1/YKL-40, GAL3, ANG2, IP-10, IL-10, TNFα, IL-6, soluble gp130, soluble IL-6R were quantified in plasma samples using Luminex assays. The Mann-Whitney U test, the Kruskal-Wallis test, correlation and regression analyses were performed. Mediation analyses were used to investigate the possible causal relationships between cytokines, lung damage, and outcomes. AVIEW lung cancer screening software, pyradiomics, and XGBoost classifier were used for radiomic feature analyses. RESULTS: CCL18, CHI3L1, and ANG2 systemic levels mainly reflected the extent of lung injury. Increased levels of every cytokine, but particularly of IL-6, were associated with the three outcomes: hospitalization, mechanical ventilation, and death. Soluble IL-6R showed a slight protective effect on death. The effect of age on COVID-19 outcomes was partially mediated by cytokine levels, while CT scores considerably mediated the effect of cytokine levels on outcomes. Radiomic-feature-based models confirmed the association between lung imaging characteristics and CCL18 and CHI3L1. CONCLUSION: Data suggest a causal link between cytokines (risk factor), lung damage (mediator), and COVID-19 outcomes.

Treatment of giant cell arteritis with ultra-short glucocorticoids and tocilizumab: the role of imaging in a prospective observational study.

OBJECTIVES: To assess the impact of tocilizumab (TCZ) monotherapy after ultra-short-pulse glucocorticoids (GCs) on clinical manifestations, and vessel inflammation and damage in large vessel-GCA (LV-GCA). METHODS: In this prospective observational study, we enrolled patients with active LV-GCA. All patients received 500 mg per day i.v. methylprednisolone for three consecutive days and weekly s.c. TCZ injections from day 4 until week 52. PET/CT was performed on all patients at baseline and at weeks 24 and 52. The primary end points were the reduction in the PET vascular activity score (PETVAS) at weeks 24 and 52 compared with baseline, and the proportion of patients with relapse-free remission at weeks 24 and 52. The secondary end point was the proportion of patients with new aortic dilation at weeks 24 and 52. RESULTS: A total of 18 patients were included (72% female, mean age 68.5 years). Compared with the baseline value, a significant reduction in the PETVAS was observed at weeks 24 and 52, mean (95% CI) reductions -8.6 (-11.5 to -5.7) and -10.4 (-13.6 to -7.2), P = 0.001 and 0.002, respectively. The proportion of patients with relapse-free remission at weeks 24 and 52 was 10/18 (56%, 95% CI 31-78) and 8/17 (47%, 95% CI 23-72), respectively. At weeks 24 and 52, no patient had shown new aortic dilation. However, 4 patients who had shown aortic dilation at baseline showed a significant increase in aortic diameter (≥5 mm) at week 52. CONCLUSION: TCZ monotherapy after ultra-short-pulse GCs controlled the clinical symptoms of GCA and reduced vascular inflammation. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT05394909.

Senescent cells in giant cell arteritis display an inflammatory phenotype participating in tissue injury via IL-6-dependent pathways.

OBJECTIVES: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. To address this question, we applied an established senescence specific multimarker algorithm in temporal artery biopsies (TABs) of GCA patients. METHODS: 75(+) TABs from GCA patients, 22(-) TABs from polymyalgia rheumatica (PMR) patients and 10(-) TABs from non-GCA/non-PMR patients were retrospectively retrieved and analysed. Synovial tissue specimens from patients with inflammatory arthritis and aorta tissue were used as disease control samples. Senescent cells and their histological origin were identified with specific cellular markers; IL-6 and MMP-9 were investigated as components of the senescent associated secretory phenotype by triple costaining. GCA or PMR artery culture supernatants were applied to fibroblasts, HUVECs and monocytes with or without IL-6R blocking agent to explore the induction of IL-6-associated cellular senescence. RESULTS: Senescent cells were present in GCA arteries at higher proportion compared with PMR (9.50% vs 2.66%, respectively, p<0.0001) and were mainly originated from fibroblasts, macrophages and endothelial cells. IL-6 was expressed by senescent fibroblasts, and macrophages while MMP-9 by senescent fibroblasts only. IL-6(+) senescent cells were associated with the extension of vascular inflammation (transmural inflammation vs adventitia limited disease: 10.02% vs 4.37%, respectively, p<0.0001). GCA but not PMR artery culture supernatant could induce IL-6-associated senescence that was partially inhibited by IL-6R blockade. CONCLUSIONS: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the tissue inflammatory bulk, suggesting a potential implication in disease pathogenesis.

Effectiveness and safety of a 26-week taper regimen of glucocorticoid in GCA patients: Results from a prospective cohort study.

OBJECTIVES: To assess the effectiveness and safety of the 26-week tapering regimen of glucocorticoids (GC) used in the GiACTA trial in a prospective cohort of treatment-naive, biopsy-proven GCA patients. METHODS: Patients with a new diagnosis of biopsy-proven GCA enrolled in the GC arm of the START project (molecular stratification of patients with GCA to tailor GC and tocilizumab therapy) were included. All patients were treated with the 26-week taper regimen of GC used in the GiACTA trial. The primary endpoint was the rate of relapse-free remission at week 52. The secondary endpoints were the proportion of patients with incident aortic damage, cumulative GC doses and GC-related adverse events (AE). RESULTS: 22 patients were included between December 2018 and February 2022. At week 52, 10 patients (45 %, 95 % CI 24-68) were in relapse-free remission. After a median (IQR) follow-up of 35 (22-40) months, 7 patients (32 %, 95 % CI 14-55) were in relapse-free remission. 18 patients with baseline large-vessel imaging underwent CT angiography at the end of the follow-up. No patients had evidence of new aortic dilation, significant progression of aortic damage or large vessel stenosis. 15/22 patients (68 %) had at least one relapse during follow-up. No patients developed visual or cerebrovascular manifestations during relapses. 15/22 (68 %) patients had at least one GC-related AE. CONCLUSIONS: A 26 week taper regimen of GC was effective and safe in inducing and maintaining remission in a sizeable proportion of newly diagnosed GCA patients. However, the frequency of GC-related adverse events was high.

C-reactive protein gene polymorphisms influence susceptibility and outcomes of biopsy-proven giant cell arteritis in Italian patients.

OBJECTIVES: To investigate potential associations between the two functional C-reactive protein (CRP) gene polymorphisms at position 3872C>T (rs1205) and 4741G>C (rs3093068) and susceptibility, clinical expression, laboratory and pathological findings, and outcomes of giant cell arteritis (GCA) in a Nothern Italian population. METHODS: One hundred and seventy Italian patients with biopsy-proven GCA resident in Reggio Emilia area, Italy, and 200 healthy controls from the same geographic area were genotyped for rs1205 and rs3093068 CRP gene polymorphisms by molecular methods. The patients were subgrouped on the basis of the presence or absence of clinical manifestations, histological and laboratory findings, and outcomes. RESULTS: The distribution of rs1205 genotype was significantly different between GCA patients and controls (p=0.018). Homozygosity for T allele was significantly more frequent in GCA patients compared to controls [p=0.006; odds ratio (OR): 2.28 (95% CI: 1.1, 4.8)]. The distribution of rs3093068 genotype differed significantly between GCA patients and controls (p=0.010). Allele C and the carriers of the C allele (C/C+C/G) of rs3093068 genotype were significantly less frequent in GCA patients compared to controls [p=0.002, OR: 0.39 (95% CI: 0.24-0.73); p=0.002, OR: 0.35 (95% CI: 0.17-0.70), respectively]. No significant associations were found between the two polymorphisms and baseline clinical manifestations. The carriers of the allele C of rs3093068 genotype had significantly higher CRP values at diagnosis (13.2±5.0 vs. 8.3±6.0 mg/dl, p=0.007). Homozygosity for T allele of rs1205 genotype had a significantly more frequent eosinophil infiltration of the temporal artery wall (21.4% vs. 6.0%) (p=0.010, OR 4.28;1.31-13.98) than patients carrying the allele C. Carriers of the allele T of rs1205 genotype had lower glucocorticoid (GC) treatment duration (p=0.041), lower cumulative total GC dose (p=0.017), and higher prevalence of long-term remission (p=0.024). CONCLUSIONS: CRP gene rs1205 and rs3093068 polymorphisms influence GCA susceptibility and its outcomes.

Artificial Intelligence-Assisted Processing of Anterior Segment OCT Images in the Diagnosis of Vitreoretinal Lymphoma.

Anterior segment optical coherence tomography (AS-OCT) allows the explore not only the anterior chamber but also the front part of the vitreous cavity. Our cross-sectional single-centre study investigated whether AS-OCT can distinguish between vitreous involvement due to vitreoretinal lymphoma (VRL) and vitritis in uveitis. We studied AS-OCT images from 28 patients (11 with biopsy-proven VRL and 17 with differential diagnosis uveitis) using publicly available radiomics software written in MATLAB. Patients were divided into two balanced groups: training and testing. Overall, 3260/3705 (88%) AS-OCT images met our defined quality criteria, making them eligible for analysis. We studied five different sets of grey-level samplings (16, 32, 64, 128, and 256 levels), finding that 128 grey levels performed the best. We selected the five most effective radiomic features ranked by the ability to predict the class (VRL or uveitis). We built a classification model using the xgboost python function; through our model, 87% of eyes were correctly diagnosed as VRL or uveitis, regardless of exam technique or lens status. Areas under the receiver operating characteristic curves (AUC) in the 128 grey-level model were 0.95 [CI 0.94, 0.96] and 0.84 for training and testing datasets, respectively. This preliminary retrospective study highlights how AS-OCT can support ophthalmologists when there is clinical suspicion of VRL.

Takayasu arteritis and large-vessel giant cell arteritis in Italian population. Comprehensive analysis from a single institutional cohort of 184 cases.

OBJECTIVE: To compare clinical and imaging characteristics in patients with Takayasu arteritis (TAK) and large vessel-giant cell arteritis (LV-GCA) in an Italian population. METHODS: We conducted a retrospective monocenter study comparing characteristics and outcomes of a cohort of 59 patients with TAK and a cohort of 127 patients with LV-GCA diagnosed between 1996 and 2016. Most of them (92%) were followed up for at least 24 months at Reggio Emilia Hospital (Italy). We also reviewed the literature discussing the results of the published manuscripts comparing LV-GCA to TAK RESULTS: LV-GCA patients had a higher prevalence of males (p = 0.003), and more frequently presented with cranial symptoms (p = 0.001), fever ≥38 °C (p = 0.007), polymyalgia rheumatica (p = 0.001), and hypertension (p = 0.001), and they had higher ESR levels at diagnosis (p = 0.0001). Differently, TAK patients had longer delay to diagnosis from the beginning of symptoms (p = 0.048), they presented more frequently with loss of pulses of large arteries (p = 0.0001), vascular bruits (p = 0.001), limb claudication (p = 0.003), myocardial infarction/angina (p = 0.03), and hypertension induced by renal artery stenosis (p = 0.001). Regarding treatment, TAK patients received a higher total and at 1 year cumulative prednisone doses (p = 0.0001 and p = 0.001, respectively), they had a longer duration of prednisone therapy (p = 0.008), and received during follow-up more frequently traditional immunosuppressants (p = 0.0001) and biological agents (p = 0.0001). Flares were more frequently observed in TAK patient (p = 0.001), while no differences were observed for long-term remission. New vascular procedures during the follow-up were more frequently performed in TAK patients (p = 0.0001). Regarding imaging at diagnosis, TAK patients had more frequently vascular stenosis/occlusion (p = 0.0001) and a higher number of vessels with structural damage per person (p = 0.0001), while LV-GCA patients had a higher number of inflamed vessels per person (p = 0.0001). Comparing the involved vascular districts at diagnosis for the presence of vessel inflammation and/or arterial damage, patients with LV-GCA had a more frequent involvement of thoracic and abdominal aorta (p = 0.024 and p = 0.007, respectively), and axillary, iliac and femoral arteries (p = 0.018, p = 0.002, and p = 0.0001. respectively), while in TAK patients, brachiocephalic, celiac, mesenteric and renal arteries were more frequently involved (p = 0.011, p = 0.019, p = 0.019, and p = 0.005, respectively). At imaging arterial damage at diagnosis was more frequently observed in TAK patients, specifically at common carotid, brachiocephalic, and subclavian arteries (p = 0.0001, p = 0.006, p = 0.0001, respectively) and descending aorta (p = 0.022). Regarding imaging during the follow-up, TAK patients developed more frequently new vascular stenosis/occlusion (p = 0.0001) and new vascular thickening (p = 0.002), no differences were observed for the development of new dilatation/aneurysm between the two vasculitides. CONCLUSION: Patients with TAK and LV-GCA show a number of similarities and also differences. Indeed, it is unclear whether they are part of the same disease spectrum or they are different conditions. As more information regarding the pathogenesis and etiology becomes known, answers to these questions are like to be forthcoming.

Complement activation predicts negative outcomes in COVID-19: The experience from Northen Italian patients.

Coronavirus disease 19 (COVID-19) may present as a multi-organ disease with a hyperinflammatory and prothrombotic response (immunothrombosis) in addition to upper and lower airway involvement. Previous data showed that complement activation plays a role in immunothrombosis mainly in severe forms. The study aimed to investigate whether complement involvement is present in the early phases of the disease and can be predictive of a negative outcome. We enrolled 97 symptomatic patients with a positive RT-PCR for SARS-CoV-2 presenting to the emergency room. The patients with mild symptoms/lung involvement at CT-scan were discharged and the remaining were hospitalized. All the patients were evaluated after a 4-week follow-up and classified as mild (n. 54), moderate (n. 17) or severe COVID-19 (n. 26). Blood samples collected before starting any anti-inflammatory/immunosuppressive therapy were assessed for soluble C5b-9 (sC5b-9) and C5a plasma levels by ELISA, and for the following serum mediators by ELLA: IL-1ß, IL-6, IL-8, TNFα, IL-4, IL-10, IL-12p70, IFNγ, IFNα, VEGF-A, VEGF-B, GM-CSF, IL-2, IL-17A, VEGFR2, BLyS. Additional routine laboratory parameters were measured (fibrin fragment D-dimer, C-reactive protein, ferritin, white blood cells, neutrophils, lymphocytes, monocytes, platelets, prothrombin time, activated partial thromboplastin time, and fibrinogen). Fifty age and sex-matched healthy controls were also evaluated. SC5b-9 and C5a plasma levels were significantly increased in the hospitalized patients (moderate and severe) in comparison with the non-hospitalized mild group. SC5b9 and C5a plasma levels were predictive of the disease severity evaluated one month later. IL-6, IL-8, TNFα, IL-10 and complement split products were higher in moderate/severe versus non-hospitalized mild COVID-19 patients and healthy controls but with a huge heterogeneity. SC5b-9 and C5a plasma levels correlated positively with CRP, ferritin values and the neutrophil/lymphocyte ratio. Complement can be activated in the very early phases of the disease, even in mild non-hospitalized patients. Complement activation can be observed even when pro-inflammatory cytokines are not increased, and predicts a negative outcome.

Exploring Gene Expression Profiles in Primary Central Nervous System Vasculitis.

OBJECTIVE: This study was undertaken to explore the gene expression profile of primary central nervous system vasculitis (PCNSV). METHODS: Brain specimens of 4 patients with granulomatous vasculitis (GV), 5 with lymphocytic vasculitis (LV), 4 with amyloid ß-related angiitis (ABRA), and 4 normal controls were studied. RNA-sequencing was performed using the Illumina Hiseq-4,000 platform and the Illumina TruSeq Total-RNA library. Student t test and false discovery rate tests were performed for each of the differentially expressed transcripts. Ingenuity Pathway Analysis was used for the pathway expression analysis. CIBERSORT was used to estimate the abundances of different immune cell subsets in the tissues based on gene expression data. RESULTS: Transcripts differentially expressed between PCNSV and normal brain indicated that endosomal, mitochondrial, and ribosome dysfunction, alterations in protein synthesis, and noncoding RNAs might be involved in PCNSV. Pathway analysis revealed the activation of dendritic cell maturation and antigen processing as well as neuroinflammation in PCNSV versus normal brain, whereas oxidative phosphorylation was inhibited. CIBERSORT estimation of immune cell subsets suggested that activated NK cells, M1 macrophages, memory B cells, and follicular helper T cells were likely to be more prevalent in PCNSV samples. Naïve CD4 T cells and monocytes were mainly estimated to be present in GV and ABRA. Plasma cell and γδ T-cell signatures were mainly found in LV and normal brain. GV showed higher levels of genes associated with macrophage activities and T cells. ABRA showed higher levels of long noncoding RNAs and miR-616. LV showed higher levels of genes encoding immunoglobulins. INTERPRETATION: RNA sequencing confirmed PCNSV heterogeneity. ANN NEUROL 2023;93:120-130.

Comparable cytokine release ex-vivo by whole blood from COVID-19 patients with and without non-invasive ventilation.

T cells are key players in the resolution of the infection by SARS-CoV-2. A delay in their activation can lead to severe COVID-19. The present work aimed to identify differences in cytokine release by T cells ex-vivo between COVID-19 patients in the acute phase, showing diverse disease severity. Concentrations of IFNγ, Granzyme B, IL-6, IL-10, IL-17A, IL-18, IP-10, MCP-1, and TNFα were evaluated after stimulation ex-vivo of whole blood samples with peptides from SARS-CoV-2 spike protein and a mitogen as well as without stimulation. Samples derived from hospitalized COVID-19 patients and SARS-CoV-2 vaccinated controls (CTR). Patients were classified on disease severity considering the necessity of non-invasive ventilation (NIV). Samples from patients requiring NIV revealed a similar release of cytokines compared with patients without NIV. COVID-19 patients showed higher spontaneous production of IFNγ and IP-10, lower production of MCP-1 after SARS-CoV-2 peptide stimulation and lower production of IFNγ, IL-10, IL-17A, Granzyme B, IP-10 after mitogenic stimulus compared with CTR. In conclusion, differences in T cell responses evaluated ex-vivo by a whole blood-based cytokine release assay do not appear to explain the need for non-invasive ventilation in COVID-19 patients.

Primary Vitreoretinal Lymphoma: Current Diagnostic Laboratory Tests and New Emerging Molecular Tools.

Primary vitreoretinal lymphoma (PVRL), a rare aggressive malignancy primarily involving the retina and/or the vitreous, is a major diagnostic challenge for clinicians (who commonly misdiagnose it as chronic uveitis) as well as for pathologists (for biological and technical reasons). Delays in diagnosis and treatment are responsible for visual impairments and life-threatening consequences, usually related to central nervous system involvement. The identification of lymphoma cells in vitreous fluid, obtained by vitrectomy, is required for diagnosis. Of note, the scarcity of neoplastic cells in small volumes of vitreous sample, and the fragility of lymphoma cells with degenerative changes caused by previous steroid use for presumed uveitis makes diagnosis based on cytology plus immunophenotyping difficult. Interleukin levels, immunoglobulin heavy chain or T-cell receptor gene rearrangements, and MYD88 mutation are applied in combination with cytology to support diagnosis. We aim to describe the current laboratory technologies for PVRL diagnosis, focusing on the main issues that these methods have. In addition, new emerging diagnostic strategies, such as next-generation sequencing analysis, are discussed. The genetic profile of PVRL remains largely unexplored. Better knowledge of genetic alterations is critical for precision medicine interventions with target-based treatments of this lymphoma for which no standardised treatment protocol currently exists.

Modifications of Chest CT Body Composition Parameters at Three and Six Months after Severe COVID-19 Pneumonia: A Retrospective Cohort Study.

We aimed to describe body composition changes up to 6-7 months after severe COVID-19 and to evaluate their association with COVID-19 inflammatory burden, described by the integral of the C-reactive protein (CRP) curve. The pectoral muscle area (PMA) and density (PMD), liver-to-spleen (L/S) ratio, and total, visceral, and intermuscular adipose tissue areas (TAT, VAT, and IMAT) were measured at baseline (T0), 2-3 months (T1), and 6-7 months (T2) follow-up CT scans of severe COVID-19 pneumonia survivors. Among the 208 included patients (mean age 65.6 ± 11 years, 31.3% females), decreases in PMA [mean (95%CI) -1.11 (-1.72; -0.51) cm2] and in body fat areas were observed [-3.13 (-10.79; +4.52) cm2 for TAT], larger from T0 to T1 than from T1 to T2. PMD increased only from T1 to T2 [+3.07 (+2.08; +4.06) HU]. Mean decreases were more evident for VAT [-3.55 (-4.94; -2.17) cm2] and steatosis [L/S ratio increase +0.17 (+0.13; +0.20)] than for TAT. In multivariable models adjusted by age, sex, and baseline TAT, increasing the CRP interval was associated with greater PMA reductions, smaller PMD increases, and greater VAT and steatosis decreases, but it was not associated with TAT decreases. In conclusion, muscle loss and fat loss (more apparent in visceral compartments) continue until 6-7 months after COVID-19. The inflammatory burden is associated with skeletal muscle loss and visceral/liver fat loss.