Raffaella Aldigeri

Transcriptomic Analysis of Human Polarized Macrophages: More than One Role of Alternative Activation?

Background Macrophages are a heterogeneous cell population which in response to the cytokine milieu polarize in either classically activated macrophages (M1) or alternatively activated macrophages (M2). This plasticity makes macrophages essential in regulating inflammation, immune response and tissue remodeling and a novel therapeutic target in inflammatory diseases such as atherosclerosis. The aim of the study was to describe the transcriptomic profiles of differently polarized human macrophages to generate new hypotheses on the biological function of the different macrophage subtypes. Methods and Results Polarization of circulating monocytes/macrophages of blood donors was induced in vitro by IFN-γ and LPS (M1), by IL-4 (M2a), and by IL-10 (M2c). Unstimulated cells (RM) served as time controls. Gene expression profile of M1, M2a, M2c and RM was assessed at 6, 12 and 24h after polarization with Whole Human Genome Agilent Microarray technique. When compared to RM, M1 significantly upregulated pathways involved in immunity and inflammation, whereas M2a did the opposite. Conversely, decreased and increased expression of mitochondrial metabolism, consistent with insulin resistant and insulin sensitive patterns, was seen in M1 and M2a, respectively. The time sequence in the expression of some pathways appeared to have some specific bearing on M1 function. Finally, canonical and non-canonical Wnt genes and gene groups, promoting inflammation and tissue remodeling, were upregulated in M2a compared to RM. Conclusion Our data in in vitro polarized human macrophages: 1. confirm and extend known inflammatory and anti-inflammatory gene expression patterns; 2. demonstrate changes in mitochondrial metabolism associated to insulin resistance and insulin sensitivity in M1 and M2a, respectively; 3. highlight the potential relevance of gene expression timing in M1 function; 4. unveil enhanced expression of Wnt pathways in M2a suggesting a potential dual (pro-inflammatory and anti-inflammatory) role of M2a in inflammatory diseases.

Flares in Biopsy-Proven Giant Cell Arteritis in Northern Italy: Characteristics and Predictors in a Long-Term Follow-Up Study.

AbstractThis study evaluated the frequency, timing, and characteristics of flares in a large cohort of Italian patients with biopsy-proven giant cell arteritis (GCA) and to identify factors at diagnosis able to predict the occurrence of flares. We evaluated 157 patients with biopsy-proven transmural GCA diagnosed and followed at the Rheumatology Unit of Reggio Emilia Hospital (Italy) for whom sufficient information was available from the time of diagnosis until at least 4 years of follow-up. Fifty-seven patients (36.5%) experienced ≥1 flares. Fifty-one (46.4%) of the 110 total flares (88 relapses and 22 recurrences) were experienced during the first 2 years after diagnosis. The majority of relapses occurred with doses of prednisone ⩽ 10 mg/day (82.9%), whereas only 3.4% of relapses occurred for doses ≥ 25 mg/day. Polymyalgia rheumatica (46.5%) and cranial symptoms (41.9%) were the most frequent manifestations at the time of the first relapse. Cumulative prednisone dose during the first year and total cumulative prednisone dose were significantly higher in flaring patients compared with those without flares (7.8 ± 2.4 vs 6.7 ± 2.4 g, P = 0.02; 15.5 ± 8.9 vs 10.0 ± 9.2 g, P = 0.0001, respectively). The total duration of prednisone treatment was longer in flaring patients (58 ± 44 vs 30 ± 30 months, P = 0.0001).Patients with disease flares had at diagnosis more frequently systemic manifestations (P = 0.02) and fever ≥ 38°C (P = 0.02), significantly lower hemoglobin levels (P = 0.05), more frequent presence at temporal artery biopsy (TAB) specimens of giant cells (P = 0.04) and intraluminal acute thrombosis (P = 0.007), and more moderate/severe arterial inflammation (P = 0.009) compared with those without flares. In the multivariate model fever ≥ 38 °C (hazard ratio 2.14; 95% confidence interval, 1.06–4.32, P = 0.03) and the severity of inflammatory infiltrate (moderate/severe versus mild) (hazard ratio 5.41; 95% confidence interval, 1.64–17.87, P = 0.006) were significantly associated with an increased risk of flares. In conclusion, a flaring course is common in GCA and it is associated with prolonged GC requirements. Fever at diagnosis and severity of inflammation at TAB appear to predict the development of disease flares.

Searching for viral antibodies and genome in intraocular fluids of patients with Fuchs uveitis and non-infectious uveitis

BackgroundTo characterise the polyspecific intraocular antibody synthesis in aqueous humor of patients with Fuchs uveitis and other types of non-infectious uveitis.MethodsAqueous and serum samples collected from 24 patients with Fuchs uveitis, 21 patients with non-infectious uveitis, and 27 healthy subjects undergoing elective cataract surgery (control group) were analysed. In addition, vitreous samples, collected from seven uveitis patients (five Fuchs and two panuveitis) during retinal surgery, were examined. Specific immunoglobulin G antibodies against cytomegalovirus (CMV), rubella virus, herpes simplex virus (HSV), and varicella zoster virus (VZV) were investigated, and Goldmann–Witmer coefficients (GWCs) were calculated. Real-time PCR was performed to detect viral genome for HSV, VZV, and CMV, while nested PCR was conducted to detect rubella RNA.ResultsNone of the control samples tested positive for any of the viral antibodies investigated. Intraocular antibody production was found in eight samples of patients affected by Fuchs uveitis (6/8 positive for rubella virus and 2/8 positive for herpes virus). Among patients with non-infectious uveitis, three tested positive for intraocular antibody production (one RV, one HSV and one for VZV). PCR was positive for RV in two patients with Fuchs uveitis, in three patients with non-infectious uveitis (one for RV and two for HSV), and in three control subjects (one for CMV and one for HSV).ConclusionsOur series confirmed the presence of specific viral antibodies, especially against rubella virus, in the subgroup of patients affected by Fuchs uveitis, suggesting that this virus may be responsible for this chronic inflammatory condition. Rubella virus is probably the main causative agent of Fuchs uveitis, but other viruses may also be involved in the pathogenesis of this disease.

Effects of a New Nutraceutical Formulation (Berberine, Red Yeast Rice and Chitosan) on Non-HDL Cholesterol Levels in Individuals with Dyslipidemia: Results from a Randomized, Double Blind, Placebo-Controlled Study

Increased non high-density lipoprotein (HDL)/low-density lipoprotein (LDL) cholesterol levels are independent risk factors for cardiovascular (CV) mortality with no documented threshold. A new combination of nutraceuticals (berberine 200 mg, monacolin K 3 mg, chitosan 10 mg and coenzyme Q 10 mg) with additive lipid-lowering properties has become available. The aim of the study is to test the efficacy of the nutraceutical formulation (one daily) in lowering non-HDL cholesterol vs. placebo at 12 weeks in individuals with non-HDL-cholesterol levels ≥160 mg/dL. 39 subjects (age 52 ± 11 years; 54% females; body mass index 27 ± 4 kg/m2) were randomized (3:1) in a double blind phase II placebo-controlled study. At baseline, 4 and 12 weeks main clinical/biohumoral parameters, pro-inflammatory cytokines, (gut)-hormones, proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and endothelial progenitor cell (EPC) number were assessed. Baseline characteristics were comparable in the two groups. The intervention significantly decreased non-HDL cholesterol (−30 ± 20 mg/dL; p = 0.012), LDL cholesterol (−31 ± 18 mg/dL, p = 0.011) and apolipoprotein (Apo) B (−14 ± 12 mg/dL, p = 0.030) levels compared to the placebo. Pro-inflammatory, hormonal, PCSK9 and EPC levels remained stable throughout the study in both groups. The intervention was well tolerated. Three adverse events occurred: Epstein Barr virus infection, duodenitis and asymptomatic but significant increase in creatine phosphokinase (following intense physical exercise) which required hospitalization. The tested nutraceutical formulation may represent a possible therapeutic strategy in dyslipidemic individuals in primary prevention.

G/R 241 polymorphism of intercellular adhesion molecule 1 (ICAM-1) is associated with Fuchs uveitis.

PURPOSE To investigate potential associations of the ICAM-1 gene polymorphisms and Fuchs uveitis in a cohort of Italian patients. METHODS Seventy-one consecutive Italian patients affected by Fuchs uveitis were observed at the Ocular Immunology Unit, Arcispedale S. Maria Nuova (Reggio Emilia, Italy) from 2002 to 2008. Two hundred twenty-six healthy Italian blood donors from the same geographic area were selected as the control group. All Fuchs uveitis patients and control subjects were genotyped by polymerase chain reaction (PCR) and allele-specific oligonucleotide techniques for ICAM-1 polymorphisms at codon 241 (exon 4). RESULTS The frequency of the ICAM-1 G/R 241 polymorphism was significantly higher in Fuchs uveitis than in the control subjects (16.9% vs. 5.8%; P=0.006, Pcorr=0.012; odds ratio, 3.3; 95% confidence interval, 1.4-7.7). No significant association between clinical features and ICAM-1 polymorphisms was found. CONCLUSIONS This study demonstrates for the first time that the ICAM-1 G/R 241 polymorphism may represent a candidate gene for Fuchs uveitis susceptibility.

Diagnosis and Treatment of Listeria monocytogenes Endophthalmitis: A Systematic Review

ABSTRACT Purpose: Describe patient characteristics, treatment, and vision outcomes of Listeria monocytogenes endophthalmitis, an exceedingly rare form of listeriosis. Methods: L. monocytogenes endophthalmitis cases in human adults, located through Medline (32) and from disease surveillance centers (11). L. monocytogenes conjunctivitis and keratitis were excluded. Results: Most cases occurred in 2000–2015 (22/43), and almost all in Europe or North America (40/43). Patients were a median 61 years, 57% male (24/42) and half were immunosuppressed. Median days from entering care to diagnosis was 8 (IQR = 5–17). Only four were exogenous infections. L. monocytogenes was identified in 31/35 of anterior eye fluid samples (89%). Antibiotic regimens varied markedly (mostly ≥3 drugs). At diagnosis, most were blind in the affected eye (85%, 28/33), only a third regained normal vision (12/36). Older patients had poorer outcomes. Conclusions: Cases increased over time. Diagnostic delays were common and visual impairment often refractory to treatment, especially in older adults. The condition’s rarity and variation in treatment makes it difficult to identify optimum therapy.

Aortic Dilatation in Patients with Large Vessel Vasculitis: a Longitudinal Case Control Study Using PET/CT

OBJECTIVE To evaluate aortic diameter and predictors of aortic dilatation using 18FDG-PET/CT in a longitudinally followed cohort of patients with large vessel vasculitis (LVV) compared with controls. METHODS All consecutive patients with LVV who underwent at least 2 PET/CT scans between January 2008 and May 2015 were included. The first and last PET/CT study was evaluated by a radiologist and a nuclear medicine physician. Diameter and FDG uptake of the aorta was measured at 4 different levels: ascending, descending thoracic, suprarenal and infrarenal abdominal aorta. Twenty-nine age- and sex-matched patients with lymphoma who underwent at least 2 PET/CT scans in the same time interval were selected as controls. RESULTS 93 patients with LVV were included in the study. In the time interval between first and last PET/CT study (median time 31 months), the diameter of the ascending, descending thoracic and suprarenal abdominal aorta significantly increased in LVV patients but not in controls. At last PET/CT, patients with LVV compared with controls had higher diameter of ascending [35.41 (5.54) vs 32.97 (4.11) mm, p = 0.029], descending thoracic [28.42 (4.82) vs 25.72 (3.55) mm, p = 0.007] and suprarenal abdominal aorta, mean [25.34 (7.01) vs 22.16 (3.26) mm, p = 0.005] and more frequently had aortic dilatation [19% vs 3%, p = 0.023]. Significant predictors of aortic dilatation were male sex [OR 7.27, p = 0.001] and, only for GCA, hypertension [OR 6.30, p = 0.031]. Finally, GCA patients with aortic FDG uptake grade 3 at first PET/CT, compared to those with aortic FDG uptake ≤2, had significantly higher aortic diameter. CONCLUSIONS Patients with LVV are at increased risk of aortic dilatation compared with age- and sex-matched controls. Significant predictors of aortic dilatation are male sex and, only for GCA, hypertension. GCA patients with aortic FDG uptake grade 3 are at increased risk of aortic dilatation.

Causes of referral to the first endocrine visit of patients with thyroid carcinoma in a mildly iodine-deficient area

To evaluate the causes of the first referral to an endocrine visit of patients with thyroid cancer in a mildly iodine-deficient area and to correlate them with prognostic features. We studied 298 consecutive patients (64 M and 234 F) with thyroid cancer. Of these, 281 had differentiated thyroid cancer. The causes of referral were categorized as follows: (Group A) clinical evidence of a neck lump; (Group B) incidental imaging in subjects without known thyroid diseases; (Group C) incidental imaging during a workup of thyroid disorders. Also, in differentiated thyroid cancer cases, clinical, histomorphologic, and prognostic parameters were compared among the three different groups of referral causes. In both total thyroid cancer and differentiated thyroid cancer cohorts, Group A, B, and C accounted for about 25, 35, and 40 % of causes, respectively. Considering the differentiated thyroid cancer, in Group B, ultrasound accounted for 94 % of cases, with 73 % resulting from screening or serendipitous study. Within a median follow-up of 5.6 [IQR: 2.7–9.5] years, disease-free survival was significantly lower in patients of Group A (Log-Rank test p = 0.030 vs. the other groups of causes). However, at the Cox multivariate analysis only male sex (p = 0.002) and stage (p = 0.005), but not referral cause, resulted independent predictors of events. In patients without known thyroid disease, unjustified thyroid ultrasound represents the main cause of referral of thyroid cancer patients to the first endocrine visit. The fact that this is not related to the disease-free survival strengthens the concept of the uselessness of thyroid cancer screening.

SAT0340 Distinct Distribution Patterns of Large Vessel Vasculitis Assessed with 18f-FDG PET/CT: A Cluster and Principal Component Analysis Study

Background The interpretation of data from 18F-FDG PET/CT studies on large vessel vasculitis (LVV) is difficult due to the scarcity of studies and to the heterogeneity of LVV populations examined. In the absence of gold standard parameters for the assessment of LVV arterial inflammation, there is a great need to develop analytical methodologies useful to properly exploit the information derived from such studies. Principal component analysis (PCA) and cluster analysis (CA) have not yet been applied to explore the LVV distribution patterns, assessed by 18F-FDG PET/CT. Objectives The aim of our study was to investigate whether PCA and CA are useful methods in identifying distinct distribution patterns of LVV, according to the specific diagnostic entity examined (giant cell arteritis, GCA; Takayasus arteritis, TAK). Methods A total of 130 18F-FDG PET/CT studies have been retrospectively examined by a nuclear physician blinded to clinical data. Maximum standardized uptake values (SUVmax) in 14 vascular districts including aortic segments (ascending aorta, thoracic aorta, aortic arch, discending thoracic aorta and abdominal aorta) and the main tributaries (carotid, subclavian, axillary, iliac and femoral arteries) have been measured and transformed into Z-scores. Identification of distribution patterns of vascular involvement has been performed using PCA and agglomerative hierarchical CA (SPSS Statistics, version 22nd). Results Three groups of vascular districts with similar trends in terms of standardized SUVmax have been identified: epiaortic arteries; aortic arch and ascending aorta; descending and abdominal aorta, with iliac and femoral arteries. A component including the entire aortic district was identified in TAK group, but not in GCA group. Conclusions PCA and CA approach revealed a subtle skewing in terms of distribution patterns of arterial involvement between TAK and GCA, assessed by 18F-FDG PET/CT SUVmax values. The influence of atherosclerosis and immunosenescence on the different trends in the aorta districts of TAK and GCA needs to be further elucidated. References Grayson PC, et al. Distribution of arterial lesions in Takayasus arteritis and giant cell arteritis. Ann Rheum Dis 2012;71:1329–1334. Arnaud L, et al. Cluster analysis of arterial involvement in Takayasu arteritis reveals symmetric extension of the lesions in paired arterial beds. Arthritis Rheum 2011;63:1136–40. Disclosure of Interest None declared

FRI0262 The Role of 18F-FDG-PET/CT in the Diagnosis and Follow-Up of Large Vessel Vasculitis

Background PET/CT has been proposed as a useful tool to diagnose and monitor activity in the large-vessel vasculitides (LVV) giant cell arteritis (GCA) and Takayasus Arteritis (TAK), but the best approach to evaluate vascular uptake still remains debated. Both GCA and TAK often present with non-specific symptoms and laboratory test results. 18F-FDG PET/CT (PET/CT) has shown its potential to diagnose LVV earlier than any other traditional imaging techniques. Objectives The aim of this study was to investigate the role of PET/CT in assessing disease activity in patients with LVV, and to compare different semi-quantitative vascular FDG grading approaches. Methods 28 Patients with LVV underwent a total of 135 PET/CT scans and were compared to 29 matched controls. All PET/CT scans were reviewed by a nuclear physician blinded to clinical data. A semi-quantitative analysis using the maximum and mean standardized uptake value (SUVmax, SUVmean), was performed. Vascular to liver, to spleen, to blood pool, and to muscle ratios, were, respectly, determinated, to minimize the effects of time from injection to acquisition, and of blood glucose level. PET/CT findings were correlated to the clinical indices ITAS (Indian TAK activity score) and Kerr/National Institute of Health (Kerr/NIH), as well as to serum acute-phase reactants levels (erythrocyte sedimentation rate [ESR] and C-reactive protein [CRP]). Results Arterial SUVmax was significantly higher in LVV (both active and inactive) patients than in controls in the following vascular districts: carotid (p 0.015-0.046), subclavian (p=0.035) arteries, thoracic (ascending, arch and descending) and abdominal aorta (p 0.0001-0.003). Arterial to liver SUVmax ratio (SUVmaxAorta-Liver ratio), arterial to spleen SUVmax ratio (SUVmaxArterial-Spleen ratio), and arterial to venous blood pool SUVmax ratio (SUVmaxArterial-Blood pool ratio) discriminated active from inactive LVV better than the arterial to muscular (gluteal) ratio (SUVmaxArterial-Muscle ratio). In particular SUVmaxAscending Aorta - Liver ratio with a cut off value of 0.65, provided a diagnostic sensitivity of 76% and a specificity of 79% (AUC =0.791). Using a cut-off value of 0.9 for the highest SUVmaxDescending Aorta - Liver ratio, it was possible to discriminate between active and inactive TAK patients with a sensitivity of 70% and a specificity of 92% (AUC =0.817). In GCA a cut-off value of 0.9 for SUVmaxAortic Arch-Spleen ratio, SUVmaxDescending Aorta-Spleen ratio, SUVmaxAortic Arch-Liver ratio, SUVmaxDescending Aorta–Liver ratio was able to discriminate active from inactive patients with a sensitivity of 70% and a specificity of 87% (AUC =0.84-0.87). Conclusions PET/CT is a useful tool for diagnosis and follow-up of LVV. In particular, SUVmaxDescending Aorta-Liver ratio in Takayasu, and SUVmaxAorta-Liver ratio, SUVmaxAorta-Spleen ratio, and SUVmaxAorta-Blood Pool ratio in GCA appeared the most useful tools in monitoring the disease activity during the follow-up. Disclosure of Interest None declared