Four mycobacteriophages Pisa1, Pisa4, Florence1 and Florence4, taxonomically classified as new species, were isolated using Mycobacterium smegmatis mc2155 as a host strain. Genomic analysis revealed dsDNA genomes devoid of virulence or antibiotic-resistance genes. All encoded lysogeny-associated elements, but Pisa4 lacked the integrase gene and showed a partial immunity repressor gene, suggesting a potentially impaired lysogenic cycle. Phages exhibited siphoviral morphology, short replication cycles (≤30 min) and burst sizes of 50-150 PFUs/bacterial cell. All phages remained active between pH 4-12 and up to 45 °C. Given its potentially impaired lysogeny, Pisa4 was considered a promising candidate for antibacterial applications; therefore, its interaction with THP-1 macrophages was investigated. The phage was internalized in THP-1 and co-localized with its host both after co-incubation with bacteria prior to infection and upon post-infection administration. However, phage replication was restricted to the co-incubation condition. Importantly, Pisa4 did not trigger neutrophil extracellular trap release and elicited minimal cytokine responses in peripheral blood mononuclear cells, with no TNF-α or IFN-γ induction. In contrast, a significant increase in IL-6 production was observed at high phage concentrations, which may be influenced by co-purified bacterial components. Finally, Pisa1 and Pisa4 infected 12 and 8 out of the 48 Mycobacterium abscessus clinical isolates tested, respectively, while Florence phages were inactive. Collectively, these results highlight that Pisa phages, particularly Pisa4, are promising candidates for future therapeutic development.

The production of antibodies against viral structural proteins such as the spike (S) and the nucleocapsid (N) is a hallmark of SARS-CoV-2 infection. The N protein contains several immunogenic regions. In this work we analysed epitope specificity and avidity of anti-N antibodies in COVID-19 patients. Eighty-nine COVID-19 patients were recruited. IgG, IgA, IgM antibodies to recombinant N protein and to 6 different peptides containing predicted B epitopes were measured by ELISA. Antibody avidity was evaluated by chaotropic ELISA. Anti-N IgG, IgA, and IgM were detected in 59

Background and aim Systemic Lupus Erythematosus (SLE) and Lupus Nephritis (LN) are prototypes of autoimmune diseases. Mechanisms determining the renal evolution in SLE patients and the identification of predictive biomarkers remain an open issue. Methods We reviwed data on serum levels of potential factors involved in SLE pathogenesis (i.e., NETs and dsDNA degradation) and autoantibodies associated with renal pathology (anti-dsDNA, anti-Histone 2 A and 3, anti-C1q, anti-ENO1, anti-ANXA1, anti-SOD2 IgG2) in patients recruited within the Italian collaborative Zeus study at the onset of renal symptoms (T0) and after 12 months (T12). Results Clustering analysis based on a few serum parameters allowed the stratification of LN/SLE/controls into well-separated groups. High dsDNA degradation and high anti-ENO1 antibody levels contributed to stratifying SLE and LN, respectively. Anti-ENO1, anti-SOD2, and anti-H2 A/-H3 antibodies paralleled proteinuria levels during the follow up (high at T0 and normalized after 12 months), whereas anti-dsDNA and anti-C1q IgG2 remained high for the whole observation period. Hazard Risk regression analysis indicated that anti-ENO1 and anti-H2 A IgG2 were associated with proteinuria (>1 g) and renal failure (eGFR <60 mL/min) (HRs and 95% CI highly significant). Conclusions Anti-ENO1 and anti-Histones 2A serum levels identify LN patients at the onset of renal symptoms and decrease following response to therapies. Both antibodies are associated with proteinuria and renal function loss. Our data support their use as predictive biomarkers for LN follow-up.

Background: KDM2A, a histone demethylase involved in chromatin remodeling and transcriptional regulation, has been implicated in tumorigenesis and immune modulation. Epigenetic determinants, such as H3K27me3/HeK36me3 have emerged as interesting prognostic factors, although their role in hepatocellular carcinoma (HCC) biology and its potential as a circulating biomarker remain largely unexplored. Aim: To investigate KDM2A levels in tumor tissue, circulating exosomes, plasma of HCC patients, and to assess its association with clinical and pathological features, including tumor grade and disease etiology.Materials and Methods: Immunohistochemistry (IHC) was used to evaluate protein expression in 42 HCC samples of different histological grades. Quantitative PCR (qPCR) was performed on a subgroup of HCCs (n = 10) and matched peritumoral tissues to assess KDM2A mRNA expression in FFPE samples. Exosomes were isolated from HCC plasma samples (n = 17) using a commercial isolation kit. Exosomal KDM2A cargo was evaluated: mRNA levels were quantified by RT-qPCR and normalized to a pool of healthy subjects (n = 9, HS), and KDM2A protein was detected by Western blot analysis. Circulating free KDM2A protein levels were measured using a commercial ELISA kit in plasma samples from 20 HCC patients and 15 HS.Results: At the tissue level, KDM2A protein expression increased significantly with tumor grade, from 3.2 ± 1.6% in low-grade (G1) to 59.1 ± 2.2% in high-grade (G3) HCCs (p<0.001). This grading-dependent pattern was confirmed by mRNA quantification from FFPE samples. Exosomal KDM2A mRNA levels were significantly higher in HCC patients compared with healthy controls (p < 0.01), with up to 40-fold increases in specific subgroups. Among the clinical variables analyzed, KDM2A levels significantly discriminated viral from non-viral HCC etiologies - p<0.01- (ROC p = 0.0236; AUC > 0.75; cutoff > 4.215; sensitivity = 66.7%, specificity = 81.8%). No KDM2A exosomal protein was detected in HCC and HS samples. Plasma free KDM2A protein levels were also elevated in HCC patients, showing a trend toward significance (p = 0.057) compared to HC.Conclusion: KDM2A is consistently upregulated in HCC at both circulating and tissue levels, with expression correlating with tumor grading. These preliminary results suggest that KDM2A might serve as a valuable biomarker for HCC detection and progression monitoring, highlighting its potential involvement in the epigenetic regulation of hepatocarcinogenesis, particularly in viral-related tumors, in line with previous mechanistic evidence (PMID: 37845585). Further functional and longitudinal studies are warranted to clarify KDM2A role as a diagnostic and therapeutic target.

Autoimmune diseases are conditions characterized by aberrant B-cell and T-cell reactivity against self-antigens. Autoantibodies are serological biomarkers of autoimmune diseases, as such, autoantibody testing is a key step for diagnosing and classifying many autoimmune diseases, as well as monitoring disease activity and devising a treatment strategy. Considering the rising number of people affected by autoimmune diseases worldwide, it is even more important to have efficient techniques that combine high sensitivity and specificity with reduced sample processing times and an automated high-throughput workflow. In this context, the identification and validation of new autoantigens and autoantibodies, together with the implementation of technological advancements, has led, in the last decades, to an improvement in patient diagnosis and stratification. Here, we review the major antigens of some of the most common autoimmune diseases, and the most widely used assays employed in diagnostic laboratories for the detection of their cognate antibody, confronting more traditional platforms with emerging ones in selected cases of study.

Rheumatoid arthritis (RA) associated interstitial lung disease (ILD) is the most common pulmonary manifestations of RA, with a progressive course and a poor survival. An early detection and better treatment is essential to improve outcome. We evaluated 16 analytes that could be relevant for the development of RA ILD. In an inception cohort of 1118 early RA patients, pulmonary fibrosis (PF) were identified in 60 patients after a mean follow-up of 5.3 years using high resolution computer tomography (HRCT). As controls, 124 early RA patients without PF and 94 matched population controls without known rheumatic disease were studied. Analysis of antibodies against histones 3 and 4 derived citrullinated peptides (CitH3/H4), and cytokines/chemokines levels were performed in plasma samples collected at RA diagnosis using in-house ELISA and Luminex analysis. Anti-CitH3(114–135) antibodies were the only antibody with increased frequency and levels in patients with PF versus without PF. The highest OR for PF development were found when combining positivity for anti-CitH3(114–135) and -CitH4(31–50) antibodies, OR 2.26. Levels of IL1α, IL1ß, TNFα, VEGFA and MIPα remained significantly elevated in patients with PF compared without PF, after adjustments and Bonferroni corrections. Several of the cytokines/chemokines correlated significantly with the histone antibodies in patients without PF. Partial least squares discriminant analysis including antibodies against citrullinated histon peptides and cytokines/chemokines identified significantly in PF in non-smokers. Antibodies against CitH3 peptides and several of the analysed cytokines/chemokines in samples collected at diagnosis were associated with subsequent delevopment of PF in patients with RA.

Neutrophil extracellular traps (NETs) are web-like structures composed of chromatin and proteins from neutrophil granules. Several studies highlight the heterogeneity of NETs, underscoring the challenges associated with their detection. In patients with COVID-19, high levels of NET fragments, called NET remnants, are detected in the circulation but also in alveoli and bronchioles. NET remnants are usually measured as complexes of DNA and myeloperoxidase (DNA−MPO). Taking advantage of proteomic data on NET composition, we developed new solid-phase assays to detect NET remnants, measuring complexes of DNA with alpha enolase (DNA−eno) or calprotectin (DNA−cal). The two assays were compared with the DNA−MPO test for the detection of in vitro-generated NET and serum NET remnants; all of them showed similar sensitivity in the detection of in vitro-generated NET. In an analysis of 40 patients with severe COVID-19 and 25 healthy subjects, the results of the three assays were highly correlated, and all detected significantly higher levels of NET remnants in patient sera. Moreover, the level of NET remnants correlated with impaired gas exchange and increased with the progressive decline of pulmonary function. The proposed assays thus represent a novel tool with which to evaluate NETosis; using antibodies to different NET constituents may allow their fingerprinting in different disorders.

Deposition of autoantibodies in glomeruli is a key factor in the development of lupus nephritis (LN). For a long time, anti-dsDNA and anti-C1q antibodies were thought to be the main cause of the kidney damage. However, recent studies have shown that the list of autoantibidies that have renal tropism and deposit in the kidney in LN is increasing and the link between anti-dsDNA and renal pathology is weak due to potential confounders. Aspecific bindings of dsDNA with cationic antibodies and of anti-dsDNA with several renal antigens such as actinin, laminin, entactin, and annexinA2 raised doubts about the specific target of these antibodies in the kidney. Moreover, the isotype of anti-dsDNA in SLE and LN has never received adequate interest until the recent observation that IgG2 are preponderant over IgG1, IgG3 and IgG4. Based on the above background, recent studies investigated the involvement of anti-dsDNA IgG2 and of other antibodies in LN. It was concluded that circulating anti-dsDNA IgG2 levels do not distinguish between LN versus non-renal SLE, and, in patients with LN, their levels do not change over time. Circulating levels of other antibodies such as anti-ENO1 and anti-H2 IgG2 were, instead, higher in LN vs non-renal SLE at the time of diagnosis and decreased following therapies. Finally, new classes of renal antibodies that potentially modify the anti-inflammatory response in the kidney are emerging as new co-actors in the pathogenetic scenario. They have been defined as 'second wave antibodies' for the link with detoxifying mechanisms limiting the oxidative stress in glomeruli that are classically stimulated in a second phase of inflammation. These findings have important clinical implications that may modify the laboratory approach to LN. Serum levels of anti-ENO1 and anti-H2 IgG2 should be measured in the follow up of patients for designing the length of therapies and identify those patients who respond to treatments. Anti-SOD2 could help to monitor and potentiate the anti-inflammatory response in the kidney.

Hidradenitis suppurativa (HS) is a chronic skin disease, characterized by clinical inflammation of the hair follicle with the recurrence of abscesses, nodules, and tunnels. Recently, several studies suggested a role of IL-1 family (IL-1F) cytokines in eliciting and sustaining the disease. The aim of this work is to perform a comprehensive analysis of IL-1F cytokines, soluble inhibitors and receptors in a cohort of HS patients not treated with biological agents. Sixteen patients affected by HS and 16 healthy controls were recruited; clinical data were collected and disease severity evaluated by means of the International HS Severity Score System (IHS4). Serum levels of IL-1F cytokines, inhibitors and receptors were measured using a Multiplex Assays. IL-18 and free IL-18 levels were significantly higher in patients vs controls. Among soluble inhibitors, IL-1Ra, IL-1R2 and ST2/IL-1R4 were significantly increased. IL-18, free IL-18 and IL-33 levels are strongly correlated with IHS4. Also the inhibitors IL-1Ra and IL-18BP show a correlation with IHS4. The data obtained in this study confirm the involvement of IL-1F cytokines in mediating the disease and determining its severity and suggest a possible role for IL-18 as novel serum biomarker of active disease.

Atezolizumab/Bevacizumab (AtezoBv) is a reference EMA-approved combination superior to sorafenib in aHCC. Nonetheless, primary resistance rate is as high as 20% and no circulating biomarkers of treatment benefit prediction are known. Flow-cytometry allows for a non-invasive, dynamic evaluation of peripheral immunitary status. In aHCC, it successfully stratified anti-PDL1 plus anti-CTLA4 treatment activity, whilst this is the first report of its application to AtezoBv. Consecutive aHCC patients treated with AtezoBv at Pisa Medical Oncology were prospectively enrolled from March 2021 to August 2022. Six-ml EDTA whole-blood were drawn predose at baseline (B), early on-treatment (E, cycles2/3 day1), and at first radiological evaluation (R). Four Flow-cytometry assays were designed to quantify the following cell subsets: T, B, NK, CD4+, CD8+, TIM+/LAG+/Ki67+ CD4+ and CD8+, T regulatory (TReg), classical and alternative monocytes, and neutrophils. Patients were dichotomized into primary-progressors (PP, if progressive disease was evident at first CT scan) and disease-controlled (DC, if partial response or stable disease was achieved). A cohort of healthy controls (HC) was enrolled. Distribution of immune cells concentration (cells/uL) was compared between HC and aHCC and between PP and DC with independent samples T-test or Mann-Whitney test (if normally and non-normally distributed, respectively). Cell subsets' longitudinal trend from B to E to R was compared within and among the two groups (PP and DC) with repeated measures ANOVA. Progression-free survival (PFS) and overall-survival (OS) were estimated with Kaplan Meier curves. Eleven aHCC patients (4 PP and 7 DC) and 9 HC were evaluated. AHCC cohort showed a prevalence of male gender (63.6%) and of extra-hepatic spread or macro-vascular invasion (72.7%), a median age of 68.9 (42,1-81,0) years and an even distribution between viral (HCV and/or HBV, 54.5%) and non-viral etiologies (dismetabolic or healthy livers, 45.5%). Compared to HC, aHCC patients had higher neutrophils-to-lymphocytes ratio (p=0.02), lower T (p=0.02), CD4+ (p=0.03), B (p < 0.01) and Ki67+CD8+ (p=0.05) cells, whilts higher LAG+CD4+ (p < 0.01) and LAG+CD8+ (p=0.03). Despite non-significantly different vs HC, TIM+CD4+ and TIM+CD8+ were positively correlated with LAG+ T lymphocytes (p for R2 0.04 and < 0.01, respectively) among aHCC. After treatment with AtezoBv, a DC rate of 63.7% was observed (2 PR and 5 SD) along with a PP rate of 36.3%, leading to a mPFS of 4,01 months (95% IC 2,27-10,56) and mOS of 13,03 months (95% IC 9,01-15,76; mOS for PP vs DC: 6.45 vs 15.76 months, p=0.08), at 11.09 months (95% IC 8,82-23,22) of median follow-up. Compared to DC, PP had lower NK at B (p=0.05) and E (p=0.03). Among PP, TReg increased from B to R (p=0.03), while NK increased from B to E (p=0.01), to R (p=0.03) in DC. A non-significant trend toward LAG+CD8+ decrease was seen from B to E to R in DC (3 subjects evaluable: 60.8 vs 22.1 vs 1.6 mean cells/uL). Despite hampered by low numerosity of the case-series, peripheral cells flow-cytometry identified aHCC patients as "immune down-regulated" if compared to HC, as known at tissue-level. Furthermore, NK and Treg dynamics might be a promising tool to predict AtezoBev activity.

Vaccination represents the best strategy to fight COVID-19 pandemics, especially in immune compromised subjects. In chronic lymphatic leukemia patients, a marked impairment of the immune response to mRNA SARS-CoV-2 vaccine was observed. In this report, we analyzed anti-RBD and neutralizing antibodies in CLL patients after two doses of mRNA SARS CoV 2 vaccine and evaluated the impact of Bruton kinase inhibitory agents. Twenty-seven CLL patients vaccinated with mRNA vaccines against SARS CoV-2 were recruited. Serum IgG, IgM and IgA anti-RBD antibodies and neutralizing antibodies were detected, and antibody avidity was measured. Peripheral blood leukocytes subsets were evaluated by flow cytometry. After two vaccine doses anti-RBD IgG were produced in 11/27 (40.5%) of patients and levels of IgG and IgA anti RBD in CLL patients were sensibly lower than in controls. Neutralizing antibodies were detectable in 12/27 (44.5%) of the patients and their level was lower than that observed in controls. Disease burden and treatment with Bruton kinases inhibitors markedly impaired vaccine induced antibody response. However, in responder patients, antibody avidity was comparable to normal subjects, indicating that the process of clonal selection and affinity maturation takes place as expected. Taken together, these data confirm the impact of disease burden and therapy on production of anti-RBD and neutralizing antibodies and support the current policy of vaccinating CLL patients.

BackgroundCancer patients are more vulnerable to COVID-19 and are thus given high priority in vaccination campaigns. In solid cancer patients treated with checkpoint inhibitors, we evaluated the amount of anti-RBD and neutralizing antibodies and antibody avidity after two or three doses of the vaccine.MethodsThirty-eight solid cancer patients, 15 untreated hematological patients and 21 healthy subjects were enrolled in the study. Blood was collected before the first dose (T0), 21 days after the second (T2) and in 18 solid cancer patients also 15 days after the third dose of vaccine (T3). IgG, IgM and IgA anti-RBD antibodies were detected by ELISA. Neutralizing antibodies were measured testing the inhibition of RBD binding to ACE2. Antibody avidity was evaluated in 18 patients by a urea avidity ELISA.ResultsIgG anti-RBD antibodies were produced in 65.8% of the cancer patients at T2, and in 60% of hematological patients at levels lower than healthy controls. IgM and IgA anti-RBD antibodies were also produced in 5.3% and 21% cancer patients, respectively. At T3, a significant increase in anti-RBD IgG levels was observed. Neutralizing antibodies were produced in 68.4% of cancer patients as compared with 93% of untreated hematological patients and 100% of controls, at titers lower than in healthy subjects. At T3, neutralizing antibodies and avidity of IgG anti-RBD increased; 6/18 patients negative at T2 developed neutralizing antibodies at T3.ConclusionThe data indicate that in cancer patients mRNA vaccine induces high avidity anti-RBD antibodies and neutralizing antibodies that increase after the third dose. The process of induction and selection of high-affinity antibodies is apparently unaffected by the treatment with anti-PD-1 or anti-PD-L1 antibodies.

We analyzed peripheral blood mononuclear cells (PBMCs) and serum inflammatory biomarkers in patients with mesial temporal lobe epilepsy (drug-resistant - DR, vs. drug-sensitive - DS). Patients with epilepsy showed higher levels of serum CCL2, CCL3, IL-8 and AOPP, and lower levels of FRAP and thiols compared to healthy controls (HC). Although none of the serum biomarkers distinguished DR from DS patients, when analysing intracellular cytokines after in vitro stimulation, DR patients presented higher percentages of IL-1β and IL-6 positive monocytes compared to DS patients and HC. Circulating innate immune cells might be implicated in DR epilepsy and constitute potential new targets for treatments.

CLM29 (a pyrazolo[3,4-d]pyrimidine, that inhibits RET, epidermal growth factor receptor, vascular endothelial growth factor receptor, and has an anti-angiogenic activity) has anti-neoplastic activity in papillary dedifferentiated thyroid cancer. Here we tested CLM29 in medullary thyroid cancer (MTC), in primary MTC cells (P-MTC) obtained at surgery, and in TT cells harboring (C634 W) RET mutation.CLM29 (10, 30, 50 μM) inhibited significantly (P < 0.001) the proliferation, and increased the percentage of apoptotic P-MTC, TT and human dermal microvascular endothelial cells. The inhibition of proliferation by CLM29 was similar in P-MTC cells with/without RET mutation. TT cells were injected sc in CD nu/nu mice, and tumor masses became detectable between 20 and 30 days after xenotransplantation; CLM29 (50 mg/kg/die) reduced significantly tumor growth and weight, and microvessel density. The anti-tumor activity of CLM29 has been shown in MTC in vitro, and in vivo, opening the way to a future clinical evaluation.

Background Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease which primarily affects the joints [1]. Numerous agents are currently used to modify the natural course of the disease, however, several RA patients still do not reach clinical remission[2]. A recently developed quinoxaline analogue named Rabeximod (RBM) showed clinical benefits comparable to TNF-α blockers in a mouse model of collagen antibody-induced arthritis[3]. Moreover, in a phase IIa clinical trial a significantly higher clinical efficacy of RBM + Methotrexate versus Methotrexate alone has also been confirmed. Objectives Since RBM’s exact biologic target has not been entirely established yet, our objective is to investigate its mode of action at the cellular and molecular level. Methods Two in vitro models were developed: i) Toll-like receptor (TLR)-4 and TLR-2–stimulated peripheral blood mononuclear cells (PBMCs) cultured for 24-hours and ii) Monocytes derived-macrophages (MDMs), cultured for 96-hours in persisting inflammation conditions mimicking the different phases of ACPA-positive rheumatoid synovial inflammation: recruitment, initiation, development, and persistence of the inflammatory process. PBMCs-transcriptomic analysis was performed using a NanoString nCounter SPRING Profiler (customised panel). Cytokine production was measured by ELISA. Results RBM induced dose-dependent cell mortality. Among PBMCs, monocyte viability was preferentially affected compared to lymphocytes. RBM treatment reduced IL-6 and TNF-α production both in TLR4/TLR2-stimulated PBMCs and MDMs supernatants at 24h (PBMCs) and 10-, 24-, 48- and 72-hour time-points (MDMs). RBM-induced down-regulation of IL-6 and TNF-α in PBMCs was also obtained when PBMCs were pre-incubated for 7 hours prior to TLR activation (without additional RBM), suggesting an internalisation process. After 24h, RBM induced the downregulation of several pro-inflammatory genes in TLR-4 and-2 stimulated PBMCs, including IL-6, TNF-α, CSF1, FCN1, and SPP1. Finally, after 24 hours, MDMs treated with RBM developed an altered morphology independently of the frequency of treatment exposure and the stage of inflammation. Conclusion Overall, our data suggest that RBM acts predominantly on monocytes/macrophages and can inhibit the production of pro-inflammatory molecules playing a crucial role in RA. References [1]Scherer HU, Häupl T, Burmester GR. The etiology of rheumatoid arthritis. J Autoimmun. Published online 2020. doi:10.1016/j.jaut.2019.102400 [2]Guo Q, Wang Y, Xu D, Nossent J, Pavlos NJ, Xu J. Rheumatoid arthritis: Pathological mechanisms and modern pharmacologic therapies. Bone Res. 2018;6(1). doi:10.1038/s41413-018-0016-9 [3]Hultqvist M, Nandakumar KS, Björklund U, Holmdahl R. Rabeximod reduces arthritis severity in mice by decreasing activation of inflammatory cells. Ann. Rheum Dis. 452010;69(8):1527-1532. doi:10.1136/ard.2009.121178 Acknowledgements: NIL. Disclosure of Interests Giulia Maria Ghirardi: None declared, Alessandra Nerviani: None declared, Liliane Fossati-Jimack: None declared, Elena Pontarini: None declared, Paola Italiani: None declared, Federico Pratesi: None declared, Sally Abdelmoaty Employee of: Cyxone AB, Carl Högerkorp Employee of: Cyxone AB, Costantino Pitzalis: None declared.

INTRODUCTION:Immediate and delayed hypersensitivity reactions (HSR) to COVID-19 vaccines are rare adverse events that need to be prevented, diagnosed, and managed in order to guarantee adherence to the vaccination campaign. The aims of our study were to stratify the risk of HSR to COVID-19 vaccines and propose alternative strategies to complete the vaccination.METHODS:1,640 subjects were screened for vaccinal eligibility, according to national and international recommendations. Among them, we enrolled for allergy workup 152 subjects, 43 with HSR to COVID-19 vaccines and 109 at high risk of HSR to the first dose. In vivo skin tests with drugs and/or vaccines containing PEG/polysorbates were performed in all of them, using skin prick test and, when negative, intradermal tests. In a subgroup of patients resulted negative to the in vivo skin tests, the programmed dose of COVID-19 vaccine (Pfizer/BioNTech) was administered in graded doses regimen, and detection of neutralizing anti-spike antibodies was performed in these patients after 4 weeks from the vaccination, using the SPIA method.RESULTS:Skin tests for PEG/polysorbates resulted positive in only 3% (5/152) of patients, including 2 with previous HSR to COVID-19 vaccines and 3 at high risk of HSR to the first dose. Among the 147 patients with negative skin tests, 97% (143/147) were eligible for vaccination and 87% (124/143) of them received safely the programmed COVID-19 vaccine dose. Administration of graded doses of Pfizer/BioNTech vaccine were well tolerated in 17 out of 18 patients evaluated; only 1 developed an HSR during the vaccination, less severe than the previous one, and all developed neutralizing anti-spike antibodies after 4 weeks with values comparable to those subjects who received the vaccine in unfractionated dose.CONCLUSION:On the whole, the usefulness of the skin tests for PEG/polysorbates seems limited in the diagnosis of HSR to COVID-19 vaccines. Graded doses regimen (Pfizer/BioNTech) is a safe and effective alternative strategy to complete the vaccinal course.