Publications by authors named "Mariapaola Marino"

33 Publications

Salivary Biomarkers in COVID-19 Patients: Towards a Wide-Scale Test for Monitoring Disease Activity.

J Pers Med 2021 May 8;11(5). Epub 2021 May 8.

Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario "A. Gemelli" IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.

The ongoing outbreak of coronavirus disease 2019 (COVID-19), which impairs the functionality of several organs, represents a major threat to human health. One of the hardest challenges in the fight against COVID-19 is the development of wide-scale, effective, and rapid laboratory tests to control disease severity, progression, and possible sudden worsening. Monitoring patients in real-time is highly demanded in this pandemic era when physicians need reliable and quantitative tools to prioritize patients' access to intensive care departments. In this regard, salivary biomarkers are extremely promising, as they allow for the fast and non-invasive collection of specimens and can be repeated multiple times.

Methods: We compare salivary levels of immunoglobulin A subclasses (IgA1 and IgA2) and free light chains (kFLC and λFLC) in a cohort of 29 SARS-CoV-2 patients and 21 healthy subjects.

Results: We found that each biomarker differs significantly between the two groups, with -values ranging from 10 to 10. A Receiving Operator Curve analysis shows that λFLC level is the best-suited candidate to discriminate the two groups (AUC = 0.96), with an accuracy of 0.94 (0.87-1.00 95% CI), a precision of 0.91 (0.81-1.00 95% CI), a sensitivity of 1.00 (0.96-1.00 95% CI), and a specificity of 0.86 (0.70-1.00 95% CI).

Conclusion: These results suggest λFLC as an ideal indicator of patient conditions. This hypothesis is strengthened by the consideration that the λFLC half-life (approximately 6 h) is significantly shorter than the IgA one (21 days), thus confirming the potential of λFLC for effectively monitoring patients' fluctuation in real-time.
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http://dx.doi.org/10.3390/jpm11050385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151878PMC
May 2021

Serum and urine free light chains measurements in patients with systemic sclerosis: novel biomarkers for disease activity.

Clin Exp Immunol 2021 May 1. Epub 2021 May 1.

Area Diagnostica di Laboratorio, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario 'A. Gemelli', IRCCS, Rome, Italy.

Circulating free light chains (FLCs), considered biomarkers of B cell activity, are frequently elevated in patients affected by systemic inflammatory autoimmune diseases. As the systemic sclerosis (SSc) clinical course can be variable, this study is aimed at evaluating FLCs levels in affected individuals as biomarkers of disease activity. We assessed FLC levels in serum and urine of 72 SSc patients and 30 healthy controls (HC). Results were analyzed in comparison with overall clinical and laboratory findings, disease activity index (DAI) and disease severity scale (DSS). SSc patients displayed increased levels of κ and λ FLC in serum significantly higher than HC (p = 0.0001) alongside the mean values of free κ/λ ratio and κ + λ sum (p = 0.0001). SSc patients showed increased free κ in urine with a κ/λ higher than HC (p = 0.0001). SSc patients with increased κ + λ in serum showed that erythro-sedimentation rate (p = 0.034), C-reactive protein (p = 0.003), DAI (p = 0.024) and DSS (p = 0.015) were higher if compared to SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free κ and DAI (r = 0.29, p = 0.014). In addition, SSc patients with increased free κ in urine had higher DAI (p = 0.048) than SSc patients with normal κ levels. Our results strengthen the role of serum FLC as useful biomarker in clinical practice to early diagnosis and monitor disease activity, showing for the first time that also urine FLC levels correlated with disease activity in SSc patients.
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http://dx.doi.org/10.1111/cei.13611DOI Listing
May 2021

Solving the mystery of HBV-related mixed cryoglobulinemia: potential biomarkers of disease progression.

Rheumatology (Oxford) 2021 02 16. Epub 2021 Feb 16.

Area Diagnostica di Laboratorio, Fondazione Policlinico Universitario "A. Gemelli", I.R.C.C.S, Rome, Italy.

Objectives: The biomarkers of an immunological dysregulation due to a chronic Hepatitis B virus (HBV) infection are indeed understudied. If untreated, this condition may evolve into liver impairment also co-occurred by extrahepatic involvements. Here, we aim to identify a new panel of biomarkers including IgG subclasses, Rheumatoid Factor (RF), and Free Light Chains (FLC) that may result useful and reliable for clinical evaluation of HBV-related cryoglobulinemia.

Methods: We retrospectively analyzed clinical data from 44 HBV positive patients. Patients were stratified on the presence/absence of mixed cryoglobulinemia in two group: 22 with cryoglobulins (CGs) and 22 without CGs. Samples from 20 healthy blood donors (HD) were used as negative controls. Serum samples were tested for IgG subclasses, RF (IgM, IgG, and IgA type), and FLC.

Results: We detected a strikingly different serum IgG subclasses distribution between HD and HBV positive patients together with different RF isotypes; FLC were significantly increased in HBV-patients if compared with HD while no significant difference was shown between HBV with/without MC.

Conclusion: The immune-inflammatory response triggered by HBV may be monitored by a peculiar profile of biomarkers. Our results open new perspective in precision medicine era; in these challenging times, they could be also employed to monitor the clinical course of COVID-19 patients, at high risk of HBV reactivation due to liver impairment and/or immunosuppressive therapies.
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http://dx.doi.org/10.1093/rheumatology/keab157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928620PMC
February 2021

Human Leukocyte Antigen Class II associations in late-onset Myasthenia Gravis.

Ann Clin Transl Neurol 2021 03 5;8(3):656-665. Epub 2021 Feb 5.

Dipartimento di Medicina e chirurgia traslazionale, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario "A. Gemelli" IRCCS, Sezione di Patologia generale, Rome, Italy.

Objective: Genetic factors predisposing to late-onset myasthenia gravis (LOMG) have not been clearly defined yet. However, genome-wide association studies identified Human Leukocyte Antigen (HLA) Class II alleles as a hotspot in this disease subtype. The aim of this study was to analyze the correlations of HLA Class II alleles with clinical data and titin antibodies in this patient subgroup.

Methods: This study consecutively enrolled anti-acetylcholine receptor antibody-positive, non-thymoma patients with generalized LOMG. All patients were of Italian ancestry. HLA-DRB1 and -DQB1 genotyping and serum titin antibody testing were performed in this population.

Results: A total of 107 patients (females: 28/107, 26.2%; median age of onset: 68 years, range: 50-92) were included. We found a positive association with HLA-DRB1*07 (P = 1.1 × 10 ), HLA-DRB1*14 (P = 0.0251) and HLA-DQB1*02 (P = 0.0095). HLA-DRB1*03, HLA-DRB1*11, and HLA-DQB1*03 were protective alleles (P = 7.9 × 10 , P = 0.0104, and P = 0.0067, respectively). By conditional haplotype analysis, HLA-DRB1*07-DQB1*02 was found to be the major risk haplotype (OR = 4.10; 95% C.I.: 2.80-5.99; P = 6.01 × 10 ). The mean age at onset was 73.4 years in DRB1*07 homozygotes, 69.7 years in heterozygotes, and 66.6 in non-carriers (P = 0.0488). DRB1*07 carriers and non-carriers did not differ in disease severity and response to therapy. Titin antibodies were detected in 61.4% of the cases, having no association with HLA alleles or specific clinical characteristics.

Interpretation: In our study, we identified the HLA DRB1*07-DQB1*02 haplotype as a predisposing factor for the development of generalized LOMG in the Italian population.
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http://dx.doi.org/10.1002/acn3.51309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951107PMC
March 2021

Acetylcholine receptor antibody positivity rate in ocular myasthenia gravis: a matter of age?

J Neurol 2021 May 2;268(5):1803-1807. Epub 2021 Jan 2.

Istituto di Neurologia, Università Cattolica del Sacro Cuore, Largo Agostino Gemelli, 8, 00168, Rome, Italy.

Background: Anti-acetylcholine receptor antibodies (AChR Abs) are detected in 85% of myasthenia gravis (MG) patients, at higher rates in patients with late-onset disease. AChR Ab frequency is generally thought to be much lower in ocular MG (OMG), although recent studies reported positivity rates higher than 70%. We hypothesized that the improved AChR Ab diagnostic yield in OMG could be related to an increased frequency of late-onset disease, as observed in generalized MG.

Methods: We compared OMG patients, with disease onset before or after 1998, for the age of onset, sex, presence of thymoma, immunosuppressive therapy rate, AChR Ab positivity, and follow-up duration. All patients had a follow-up ≥ 2 years. AChR Abs were tested by radioimmunoassay.

Results: The study included 133 patients. Disease onset occurred before 1998 in 54/133 cases (41%). Age of onset, the proportion of late-onset patients, and AChR Ab positivity rate were significantly increased in the more recent population. Thymoma frequency was similar in the two series. On multivariate analysis, the only variable predicting AChR Ab positivity was the age at onset ≥ 50 years (OR = 6.50, 95% CI = 2.70-15.63, p < 0.0001).

Conclusions: Our results confirm that current AChR Ab positivity in OMG may be higher than generally thought. In our population, this finding was associated with an increased frequency of late-onset cases.
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http://dx.doi.org/10.1007/s00415-020-10342-3DOI Listing
May 2021

Cryoglobulins: putative effectors of adaptive immune response.

Clin Exp Rheumatol 2021 Mar-Apr;39 Suppl 129(2):171-179. Epub 2020 Oct 29.

Department of Translational and Precision Medicine, Sapienza University of Rome, Italy.

Cryoglobulinaemia consists of circulating monoclonal and/or polyclonal immunoglobulins with rheumatoid factor (RF) activity that precipitate at temperatures <37°C. Cryoglobulinaemic syndrome, characterised by clinical signs of systemic vasculitis, is associated with chronic infection of hepatitis C virus (HCV) and might evolve in B-cell malignancies. In about one third of all HCV infection cases, serum autoantibodies are commonly found. This is probably due directly to the transformation of infected B cells but, also, indirectly, to the viral chronic stimulation of a pool of autoreactive B cells. The pattern of IgG subclasses seems to contribute to the worsening progression of HCV infection into lymphoproliferative and/or autoimmune diseases. Many evidences showed that B cells circulating in patients with HCV-associated mixed cryoglobulinaemia (MC) are profoundly abnormal; moreover, in most of cases, normal B cells are replaced by expanded clonal B cells characterized by the low expression of CD21. After viral eradication, these cells persist in circulation and their occurrence does not correlate with serum cryoglobulins nor with vasculitis response or relapse. It is probably due to the persistence of monoclonal B cells producing RF, that in course of MC can be reactivated by circulating immune complexes, highly produced during infections or tumours. Here, we aimed to review current literature focusing the pathogenesis of MC referring to specificity and immunochemical characteristics of the immunoglobulins involved in cryoprecipitation.
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May 2021

Epidemiological evidence for a hereditary contribution to myasthenia gravis: a retrospective cohort study of patients from North America.

BMJ Open 2020 09 18;10(9):e037909. Epub 2020 Sep 18.

Institute of General Pathology, Catholic University, Fondazione Policlinico Universitario "A. Gemelli"-I.R.C.C.S, Rome, Italy.

Objectives: To approximate the rate of familial myasthenia gravis and the coexistence of other autoimmune disorders in the patients and their families.

Design: Retrospective cohort study.

Setting: Clinics across North America.

Participants: The study included 1032 patients diagnosed with acetylcholine receptor antibody (AChR)-positive myasthenia gravis.

Methods: Phenotype information of 1032 patients diagnosed with AChR-positive myasthenia gravis was obtained from clinics at 14 centres across North America between January 2010 and January 2011. A critical review of the epidemiological literature on the familial rate of myasthenia gravis was also performed.

Results: Among 1032 patients, 58 (5.6%) reported a family history of myasthenia gravis. A history of autoimmune diseases was present in 26.6% of patients and in 28.4% of their family members.

Discussion: The familial rate of myasthenia gravis was higher than would be expected for a sporadic disease. Furthermore, a high proportion of patients had a personal or family history of autoimmune disease. Taken together, these findings suggest a genetic contribution to the pathogenesis of myasthenia gravis.
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http://dx.doi.org/10.1136/bmjopen-2020-037909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511637PMC
September 2020

COVID-19 and hepatic involvement: The liver as a main actor of the pandemic novel.

Scand J Immunol 2021 Mar 26;93(3):e12977. Epub 2020 Sep 26.

Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario "A. Gemelli" I.R.C.C.S, Rome, Italy.

In the natural history of SARS-CoV-2 infection, liver injury is frequent but quite mild and it is defined as any liver damage occurring during disease progression and treatment of infection in patients with or without pre-existing liver diseases. The underlying mechanisms for hepatic injury in patients with COVID-19 are still unclear but the liver damage in SARS-CoV-2 infection seems to be directly caused by virus-induced cytopathic effects. In this review, we will summarize all data of updated literature, regarding the relationship between SARS-CoV-2 infection, acute response and liver involvement. An overview will be given on liver injury, liver transplant and the possible consequences of COVID-19 in patients with pre-existing liver diseases.
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http://dx.doi.org/10.1111/sji.12977DOI Listing
March 2021

Immunological Role of IgG Subclasses.

Immunol Invest 2021 May 11;50(4):427-444. Epub 2020 Jun 11.

Area Diagnostica di Laboratorio, Fondazione Policlinico Universitario "A. Gemelli", Rome, Italy.

The loss of tolerance to self-antigens is the unequivocal "red line" of autoimmunity: both development of autoreactive T and B cells and production of polyclonal autoantibodies represent seminal keys to the pathogenesis of protean autoimmune diseases. Most of these autoantibodies are immunoglobulins G (IgG), functionally distinguished in four subclasses named IgG1, IgG2, IgG3, and IgG4, due to structural differences in the hinge and heavy chain constant regions. Different studies analyzed serum levels of IgG subclasses in the course of different disorders, showing that they might have a pathogenic role by regulating interactions among immunoglobulins, Fc-gamma receptors, and complement. To date, the mechanisms promoting different IgG subclasses distribution during the natural history of most autoimmune diseases remain somewhat unclear. Evidence from the medical literature shows that the serum IgG profile is peculiar for many autoimmune diseases, suggesting that different subclasses could be specific for the underlying driving autoantigens. A better knowledge of IgG subsets may probably help to elucidate their pathological task, but also to define their relevance for diagnostic purposes, patients' personalized management, and prognosis assessment.
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http://dx.doi.org/10.1080/08820139.2020.1775643DOI Listing
May 2021

Long-Lasting Rituximab-Induced Reduction of Specific-But Not Total-IgG4 in MuSK-Positive Myasthenia Gravis.

Front Immunol 2020 5;11:613. Epub 2020 May 5.

Istituto di Neurologia, Università Cattolica del Sacro Cuore, Rome, Italy.

The use of rituximab (RTX), an anti-CD20 monoclonal antibody (Ab), in refractory myasthenia gravis (MG) is associated with a better response in patients with Abs to the muscle-specific tyrosine kinase (MuSK) than in other MG subgroups. Anti-MuSK Abs are mostly IgG4 with proven pathogenicity and positive correlation with clinical severity. The rapid and sustained response to RTX may be related to MuSK Ab production by short-lived Ab-secreting cells derived from specific CD20 B cells. Here, we investigated the long-term effects of RTX in nine refractory MuSK-MG patients with a follow-up ranging from 17 months to 13 years. In patients' sera, we titrated MuSK-specific IgG (MuSK-IgG) and MuSK-IgG4, along with total IgG and IgG4 levels. Optimal response to RTX was defined as the achievement and maintenance of the status of minimal manifestations (MM)-or-better together with ≥ 50% steroid reduction, withdrawal of immunosuppressants, and no need for plasma-exchange or intravenous immunoglobulin. After a course of RTX, eight patients improved, with optimal response in six, while only one patient did not respond. At baseline, MuSK-IgG and MuSK-IgG4 serum titers were positive in all patients, ranging from 2.15 to 49.5 nmol/L and from 0.33 to 46.2 nmol/L, respectively. MuSK Abs mostly consisted of IgG4 (range 63.80-98.86%). RTX administration was followed by a marked reduction of MuSK Abs at 2-7 months and at 12-30 months ( < 0.02 for MuSK-IgG and < 0.01 for MuSK-IgG4). In patients with a longer follow-up, MuSK Ab titers remained suppressed, paralleling clinical response. In the patient who achieved long-term complete remission, MuSK-IgG4 was no longer detectable within 2 years, while MuSK-IgG remained positive at very low titers up to 10 years after RTX. In the patient who did not respond, MuSK-IgG and MuSK-IgG4 remained unchanged. In this patient series, total IgG and IgG4 transiently decreased ( < 0.05) at 2-7 months after RTX. The different trends of reduction between MuSK-IgG4 and total IgG4 after RTX support the view that short-lived Ab-secreting cells are the main producers of MuSK Abs. The ratio between short-lived Ab-secreting cells and long-lived plasma cells may influence the response to RTX, and B-cell severe depletion may reduce self-maintaining autoimmune reactivity.
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http://dx.doi.org/10.3389/fimmu.2020.00613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214629PMC
March 2021

Biomarkers of minimal residual disease in rituximab-treated patients with mixed cryoglobulinemia.

Biotechnol Appl Biochem 2021 Apr 17;68(2):319-329. Epub 2020 May 17.

Department of Translational and Precision Medicine, Sapienza University of Rome, Rome, Italy.

Hepatitis C virus (HCV) represents the major risk factor for mixed cryoglobulinemia (MC), a small-vessel vasculitis that may evolve into an overt B-cell non-Hodgkin's lymphoma. Here, we aimed to identify a biomarker signature for the early diagnosis of minimal residual disease (MRD). We assessed free light chains (FLCs), IgM k,and IgM λ heavy/light chain (HLC) pairs, and vascular endothelial growth factor (VEGF) in sera from 34 patients with MC vasculitis (32 HCV- and 2 HBV-related), treated with low-dose rituximab (RTX). FLCs and IgM HLCs were measured by turbidimetric assay; VEGF by an enzyme-linked immunosorbent assay. After RTX, the positive (complete + partial) clinical and laboratory responses were of 85.29% and 50%, respectively; in contrast, the mean levels of FLCs, IgM HLCs, and VEGF were substantially unaffected in most patients and still above the normal range. In those achieving a reduction of FLCs and IgM k and λ chains values within the range of normality, we found that post-treatment free λ chains and IgM k values correlated with clinical and laboratory response. Our results suggest that high levels of FLCs, IgM HLCs, and VEGF could represent the signature of "dormant" B cell clones' activity that could be very useful to identify MRD indicative of possible relapse or worsening outcome.
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http://dx.doi.org/10.1002/bab.1929DOI Listing
April 2021

Sentinel biomarkers in HCV positive patients with mixed cryoglobulinemia.

J Immunol Methods 2020 01 25;476:112687. Epub 2019 Oct 25.

Dipartimento di Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario "A. Gemelli" - I.R.C.C.S, Università Cattolica del Sacro Cuore, Rome, Italy.

Background: Infections, autoimmunity and cancer play a role as determinants of etiology in Hepatitis C virus (HCV) related mixed cryoglobulinemia (MC). Several factors of risk have been suggested as markers of pathogenesis and progression of HCV-related MC into B cell Non-Hodgkin's Lymphoma (B-NHL). Here, we evaluated IgG subclass distribution, free light chains (FLCs) and vascular endothelial growth factor (VEGF) as a new combination of biomarkers.

Methods: We measured IgG1-4 subclasses, FLCs and VEGF levels in sera 53 from HCV-related MC, in comparison with 40 sera from HCV negative patients with rheumatoid arthritis (RA) and 30 from healthy blood donors (HBD).

Results: IgG3 levels were significantly higher in HCV-MC patients with a decrement of IgG2 and IgG4; FLC levels significantly increased in both MC and RA patients' groups; serological VEGF was higher in HCV-MC patients than in HBD in correlation with k and λ levels.

Conclusion: Our results suggest that a specific IgG subclasses pattern together with raised levels of FLCs and VEGF could represent the biomarker "signature" of an inflammation multistage of acquired immune system.
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http://dx.doi.org/10.1016/j.jim.2019.112687DOI Listing
January 2020

The Laboratory Role in anti-TNF Biological Therapy Era.

Immunol Invest 2020 Apr 12;49(3):317-332. Epub 2019 Jul 12.

Istituto di Patologia Generale - Fondazione Policlinico Universitario Agostino Gemelli- IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy.

Along years, the advent of biological therapy widely modified treatment of rheumatic diseases and other disorders. However, many agents may elicit in anti-drug antibodies (ADAbs) upon consecutive infusions, with a loss of response. For the right strategy of a personalized medicine, the therapeutic monitoring of TNF-α inhibitors and ADAbs represents an important effort in diagnostic-therapeutic pathway, to improve overall patient management and favoring an appropriate clinical approach. A raising number of diagnostic tests have been designed to elucidate the efficacy and/or safety of a specific drug or class of drugs for a targeted patient's group. Our paper reviewed the current understanding of the immunogenicity of biological drugs employed in the treatment of inflammatory diseases underlying the laboratory role.
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http://dx.doi.org/10.1080/08820139.2019.1637434DOI Listing
April 2020

Free light chains and autoimmunity.

Autoimmun Rev 2019 May 4;18(5):484-492. Epub 2019 Mar 4.

Area Diagnostica di Laboratorio, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.

The study of free light chains (FLCs) has grown as area of enormous interest for many clinicians with the aim of disclosing the exact biological role and potential use of FLCs in the clinical routine. Moreover, the attention given to immunological functions of FLCs has sparked a new light into their pathogenic contribution in different chronic autoimmune-based inflammatory diseases. The release of intracellular antigens following cell death or ineffective clearance of apoptotic debris, modification of self-antigens, and molecular mimicry may trigger the production of immunoglobulins after activation and polyclonal expansion of B cells, by which FLCs are released. The discovery of polyclonal FLCs as potential biomarkers started with the observation of their increased concentrations in a variety of biological fluids related to patients with autoimmune diseases. This review deals with the use of polyclonal FLCs for identifying severity and monitoring outcome after treatment in some autoimmune diseases, namely systemic lupus erythematosus, myasthenia gravis, systemic sclerosis, rheumatoid arthritis and Sjögren's syndrome, as supported by the fact that levels of FLCs correlate with both B cell activation markers and other specific markers of disease activity. In a near future, following the evidence shown, FLCs might probably work as early prognostic markers of severity and also as indicators of response to treatment or early assessment of relapse in selected autoimmune diseases.
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http://dx.doi.org/10.1016/j.autrev.2019.03.003DOI Listing
May 2019

Serological profile of asymptomatic HCV positive patients with low level of cryoglobulins.

Biofactors 2019 May 18;45(3):318-325. Epub 2018 Dec 18.

Dipartimento di Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Agostino Gemelli, Università Cattolica del Sacro Cuore, Rome, Italy.

Clinical spectrum of hepatitis C virus (HCV)-related cryoglobulinemia varies from an asymptomatic presentation to severe vasculitis and lymphoma. A recent study in HCV-negative patients suggests that low cryoglobulins (CGs) levels are responsible for severe renal and neurological complications. The aim of this study was to identify a panel of serological biomarkers associated with low levels of CGs in HCV-positive patients. We studied a population of 79 untreated patients with chronic HCV infection: 13 naïve patients without CGs; 28 patients with asymptomatic mixed cryoglobulinemia (MC) and low levels of CGs (16/28 with polyclonal type III and 12/28 with microheterogeneous type III CGs); 38 patients with symptomatic MC and high levels of type II CGs. Serum samples were collected and examined for rheumatoid factor (RF) IgG and IgM, free light chains (FLCs) and C3 and C4 complement components. We found that RF-IgG and IgM, free k chains and k+λ were increased while C4 component was reduced, both in symptomatic and asymptomatic patients. Our results suggest that, even in absence of MC symptoms, the low levels of CGs may represent a trigger of activation for immune system in course of HCV infection. The identification of a correlated biomarkers panel could improve the clinical management of these patients and pave the way for target treatment strategies. © 2018 BioFactors, 45(3):318-325, 2019.
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http://dx.doi.org/10.1002/biof.1485DOI Listing
May 2019

Serological Immunoglobulin-Free Light Chain Profile in Myasthenia Gravis Patients.

J Immunol Res 2018 25;2018:9646209. Epub 2018 Mar 25.

Dipartimento di Medicina di Laboratorio, Fondazione Policlinico Universitario Agostino Gemelli, Università Cattolica del Sacro Cuore, Rome, Italy.

Background: Serological levels of free immunoglobulin light chains (FLCs), produced in excess of heavy chains during synthesis of immunoglobulins by plasma cells, can be considered a direct marker of B cell activity in different systemic inflammatory-autoimmune conditions and may represent a useful predictor of rituximab (RTX) therapeutic efficacy, as reported for rheumatoid arthritis and systemic lupus erythematosus. Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction with antibodies (abs) targeting the acetylcholine receptor (AChR) or the muscle-specific tyrosine kinase (MuSK), inducing muscle weakness and excessive fatigability. As MG course may be remarkably variable, we evaluated the possible use of FLCs as biomarkers of disease activity.

Subjects And Methods: We assessed FLC levels in 34 sera from 17 AChR-MG and from 13 MuSK-MG patients, in comparison with 20 sera from patients with systemic autoimmune rheumatic diseases and 18 from healthy blood donors, along with titers of specific auto-abs and IgG subclass distribution.

Results: We found a statistically significant increase in free chains in both AChR- and MuSK-MG patients, while free chain levels were increased only in AChR-MG. We also observed a significant reduction of both free and chains in 1/4 MuSK-MG patients along with specific abs titer, two months after RTX treatment.

Conclusions: From our data, FLCs appear to be a sensitive marker of B cell activation in MG. Further investigations are necessary to exploit their potential as reliable biomarkers of disease activity.
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http://dx.doi.org/10.1155/2018/9646209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889870PMC
September 2018

Different biochemical patterns in type II and type III mixed cryoglobulinemia in HCV positive patients.

Dig Liver Dis 2018 Sep 4;50(9):938-943. Epub 2018 Apr 4.

Institute of Internal Medicine, Fondazione Policlinico Universitario Agostino Gemelli - Università Cattolica del Sacro Cuore, Rome, Italy.

Background: Reversible cryoprecipitability of proteins is observed as a concomitant feature of immune complex formation. Mixed cryoglobulinemia (MC) is systemic vasculitis, associated with mixed IgM and IgG cryoglobulins (CGs) showing rheumatoid factor (RF) activity. It is frequently associated with hepatitis C virus (HCV). This study investigates the presence of IgG RF and anti-nuclear antibodies (ANA) in cryoprecipitates of patients with type III and type II MC, to understand the biochemical patterns associated with different types of MC to a greater degree.

Methods: Sera from 70 HCV untreated patients with type III or type II MC were tested by immunofixation for IgG3 and through ELISA for IgG RF. Cryoprecipitates were analysed for ANA by indirect immunofluorescence to identify specific patterns.

Results: After stratification according to MC type, the ANA patterns between type II and type III MC were statistically different. IgG3 levels and IgG-RF positivity were significantly higher in type III cryoprecipitate. We observed a higher positivity of IgG3 and a significant difference between the liver fibrosis stage, ANA and IgG-RF in the cryoprecipitate.

Conclusion: Results show a combination of biochemical markers and autoantibodies associated to mixed cryoglobulinemia; these findings could be further investigated in order to ascertain their usefulness in assessing the risk for the development of mixed cryoglobulinemia.
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http://dx.doi.org/10.1016/j.dld.2018.03.028DOI Listing
September 2018

Rituximab in myasthenia gravis: a "to be or not to be" inhibitor of T cell function.

Ann N Y Acad Sci 2018 02 25;1413(1):41-48. Epub 2018 Jan 25.

Istituto di Neurologia, Università Cattolica del Sacro Cuore, Rome, Italy.

In recent years, rituximab (RTX), a monoclonal antibody that binds the B lymphocyte membrane protein CD20, has been increasingly used for the treatment of autoimmune diseases, with the rationale of destroying pathogenic B lymphocytes and decreasing autoantibody formation. Surprisingly, RTX has also proven effective in predominantly T cell-mediated diseases, raising the question whether additional mechanisms may play roles in determining the therapeutic response. Here, we review the current literature on the effects of RTX in autoimmune diseases, with special emphasis on myasthenia gravis (MG). To elicit a complete and effective immune response, B and T lymphocytes cooperate in a loop in which they affect each other. Disruption of this cross talk has profound effects on the immune system. RTX is likely to affect the whole spectrum of B cell function, including antigen presentation, cytokine production, and T cell stimulation. In addition, as a small subset of T lymphocytes expresses CD20, its direct targeting by RTX may contribute to the therapeutic effect. Owing to its distinctive immune characteristics, MG proved to be a useful model to investigate the multifaceted implications of B cell depletion.
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http://dx.doi.org/10.1111/nyas.13562DOI Listing
February 2018

Myasthenia gravis with antibodies to MuSK: an update.

Ann N Y Acad Sci 2018 01 21;1412(1):82-89. Epub 2017 Dec 21.

Institute of General Pathology, Catholic University, Fondazione Policlinico Gemelli, Rome, Italy.

Myasthenia gravis with antibodies to the muscle-specific tyrosine kinase (MuSK MG) is a rare disease with distinctive pathogenic mechanisms and clinical features. An acute onset and predominant bulbar muscle weakness are very common and highly suggestive of the disease. On the other hand, a more indolent course, atypical ocular presentation, and signs of cholinergic hyperactivity may complicate the diagnosis. Though MuSK MG is still a severe disease, over the years we have observed a steady reduction in the rate of respiratory crisis and a significant improvement in the clinical outcome, both likely related to earlier diagnosis and timely treatment. Despite the improved management, MuSK MG patients tend to remain dependent on long-term immunosuppressive treatment and may develop permanent disabling weakness. In uncontrolled studies, B cell depletion with rituximab proved effective in most patients with refractory disease, inducing prolonged clinical responses associated with a sustained reduction of serum antibody levels. Promising results from experimental studies and case reports suggest that both 3,4-diaminopyridine and albuterol may be effective as symptomatic agents.
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http://dx.doi.org/10.1111/nyas.13518DOI Listing
January 2018

Low reliability of anti-KIR4.1 peptide auto-antibodies in multiple sclerosis patients.

Mult Scler 2018 06 26;24(7):910-918. Epub 2017 May 26.

Institute of General Pathology, School of Medicine, Università Cattolica del Sacro Cuore, Rome, Italy/Department of Laboratory Medicine, School of Medicine, Fondazione Policlinico Universitario A. Gemelli, Rome, Italy.

Background: Multiple sclerosis (MS) is an autoimmune disease for which auto-antibodies fully validated as diagnostic and prognostic biomarkers are widely desired. Recently, an immunoreactivity against the inward rectifying potassium channel 4.1 (KIR4.1) has been reported in a large proportion of a group of MS patients, with amino acids 83-120 being the major epitope. Moreover, a strong correlation between anti-KIR4.1 and anti-full-length-protein auto-antibodies titer was reported. However, this finding received limited confirmation.

Objective: Validation of the diagnostic potential of anti-KIR4.1 antibodies in 78 MS patients, 64 healthy blood donors, and 42 individuals with other neurological diseases.

Methods: Analysis of anti-KIR4.1 antibodies by enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum we produced as a new ELISA reliability control. Additionally, evaluation of reactivity against 293-T cells transiently transfected with full-length KIR4.1 by flow cytometry.

Results: We found antibodies to KIR4.1 only in 13 out of 78 (16.6%) MS patients; among these, only 2 were positive for anti-full-length KIR4.1 antibodies.

Conclusion: Employing a new reliability control and a new cytofluorometric assay, we cannot support anti-KIR4.1 auto-antibodies as a reliable biomarker in MS.
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http://dx.doi.org/10.1177/1352458517711275DOI Listing
June 2018

Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis.

PLoS One 2015 18;10(8):e0135378. Epub 2015 Aug 18.

Institute of General Pathology, School of Medicine, Università Cattolica S. Cuore, Rome, Italy.

Background: Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.

Methods: The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system.

Results: We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals.

Conclusion: Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135378PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540439PMC
May 2016

A Study of Inflammatory/Necrosis Biomarkers in the Fracture of the Femur Treated with Proximal Femoral Nail Antirotation.

Mediators Inflamm 2015 14;2015:189864. Epub 2015 May 14.

Institute of General Pathology, Catholic University, 00168 Rome, Italy ; Department of Orthopaedic Sciences and Traumatology, University Hospital Agostino Gemelli, Catholic University, 00168 Rome, Italy ; Department of Laboratory Medicine, University Hospital Agostino Gemelli, Catholic University, 00168 Rome, Italy.

Pertrochanteric fractures are common injuries in adults and source of morbidity and mortality among the elderly. Different surgical techniques were recommended for their treatment but undoubtedly they add an additional inflammatory trauma along the fracture itself. Many attempts to quantify the degree of approach-related trauma are carried out through measurements of systemic inflammatory parameters. In this study we prospectively analyzed laboratory data of 20 patients over eighty with pertrochanteric fracture of the femur treated with proximal femoral nail antirotation (PFNA). This is an excellent device for osteosynthesis because it can be easily and quickly inserted by a mini-incision providing stable fixation and early full mobilization. Serum tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and plasma creatin kinase (CK) were evaluated 1 hour preoperatively and 24 hours postoperatively. Our results show that PFNA did not induce significant increments in serum levels of inflammatory cytokines TNF-α and IL-6; CRP was elevated preoperatively in correlation with waiting time for surgery; CRP and CK showed a significant increment in the first postoperatory day; CK increment was correlated with surgical time length. We conclude that, for the markers we analyzed, PFNA shows a low biomechanical-inflammatory profile that represents an advantage over other techniques.
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http://dx.doi.org/10.1155/2015/189864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446479PMC
February 2016

T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients.

J Autoimmun 2014 Aug 4;52:113-21. Epub 2014 Jan 4.

General Pathology Institute, Università Cattolica del Sacro Cuore, Largo F. Vito, 1, 00168 Rome, Italy. Electronic address:

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5- DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.
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http://dx.doi.org/10.1016/j.jaut.2013.12.007DOI Listing
August 2014

PTPN22 and myasthenia gravis: replication in an Italian population and meta-analysis of literature data.

Neuromuscul Disord 2012 Feb 24;22(2):131-8. Epub 2011 Dec 24.

Institute of General Pathology, Catholic University, Largo Francesco Vito 1, 00168 Rome, Italy.

Polymorphisms in PTPN22 are associated with many autoimmune diseases; while rs2476601 is supposed to play a major role, other experimental data suggest that rs2488457 may be even more important. Results in myasthenia gravis are controversial. In 356 Italian myasthenic patients and 439 controls genotyped for both polymorphisms, we found that rs2476601 was not associated with myasthenia, presence of autoantibodies, thymus pathology, sex or onset age unlike previous studies on other European populations (confirmed by the present meta-analysis). On the other hand, while rs2488457 was not associated with myasthenia or thymus pathology, we found a correlation of rs2488457 with low autoantibody titers and a trend of association with a less severe disease. Both polymorphisms were in tight linkage disequilibrium in controls, not in patients. Our results suggest that SNPs in this gene different from rs2476601, and/or epigenetic interactions, could play a greater role.
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http://dx.doi.org/10.1016/j.nmd.2011.09.003DOI Listing
February 2012

Anti-acetylcholinesterase antibodies associate with ocular myasthenia gravis.

J Neuroimmunol 2010 Jan 26;218(1-2):102-6. Epub 2009 Nov 26.

Institute of General Pathology, Catholic University, 00168 Rome, Italy.

In MG, anti-AChR or anti-MuSK abs impair neuromuscular transmission. Partial inhibition of AChE can ameliorate symptoms, while a complete block causes a cholinergic blockade. We found anti-AChE abs in 115/240 MG patients, with no correlation with sex, age at onset, thymus pathology, presence of anti-AChR or anti-MuSK antibodies. We found a correlation with the ocular form of the disease, and with milder forms of MG not requiring immunosuppressants; moreover, when we considered only those patients who were off AChEI therapy, we found that ocular patients were positive for anti-AChE abs, while generalized patients were negative. According to an experimental model, we hypothesize that anti-AChE abs could contribute to ptosis through an inhibition of the sympathetic innervation of the tarsal muscle.
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http://dx.doi.org/10.1016/j.jneuroim.2009.11.004DOI Listing
January 2010

IL-6 regulates MCP-1, ICAM-1 and IL-6 expression in human myoblasts.

J Neuroimmunol 2008 May 8;196(1-2):41-8. Epub 2008 Apr 8.

Institute of General Pathology, Università Cattolica, L.go F. Vito 1, 00168 Roma, Italy.

The inflammatory reaction in autoimmune polymyositis and rejection of transplanted myoblasts is characterized by mononuclear cell infiltration. In other settings monocytes are locally recruited by an IL-6-induced IL-8-to-MCP-1 switch. IL-6, upon binding to soluble gp80 (sIL-6R), can interact with membrane-bound ubiquitously expressed gp130 and activate virtually all cells (transsignaling). We found that human myoblasts could use transsignaling to produce IL-6, MCP-1 and ICAM-1; the addition of sIL-6R, binding to IL-1beta-induced IL-6, greatly increases IL-6 production. These in vitro data support the hypothesis that locally secreted IL-6 can target monocyte chemotaxis and leukocytes trafficking through an IL-6, MCP-1 and ICAM-1 modulation.
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http://dx.doi.org/10.1016/j.jneuroim.2008.02.005DOI Listing
May 2008

IL-6-deficient mice show impaired inflammatory response in a model of myosin-induced experimental myositis.

J Neuroimmunol 2006 Jul 24;176(1-2):9-15. Epub 2006 May 24.

Institute of General Pathology, Catholic University, Largo F. Vito 1, 00168 Rome, Italy.

Inflammatory/immune reactions against muscle cells are responsible for the damage in idiopathic inflammatory myopathies. We investigated the role of IL-6, a cytokine known to contribute to local leukocyte accumulation, in a model of myosin-induced experimental myositis. After injection of rabbit myosin in CFA/pertussis toxin, normal mice develop clinically evident muscle deficit and damage, as demonstrated by myofiber necrosis and leukocyte infiltration, while IL-6-deficient mice have no clinical or histological signs of muscle damage. This study evidences that selective deficiency of IL-6 directly or indirectly hinders the local inflammatory response and its harmful effects in this model of muscle damage.
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http://dx.doi.org/10.1016/j.jneuroim.2006.03.026DOI Listing
July 2006

Identification of disease-specific autoantibodies in seronegative myasthenia gravis.

Ann N Y Acad Sci 2003 Sep;998:356-8

Institute of General Pathology, Catholic University, 00168 Rome, Italy.

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http://dx.doi.org/10.1196/annals.1254.041DOI Listing
September 2003

Clinical correlates with anti-MuSK antibodies in generalized seronegative myasthenia gravis.

Brain 2003 Oct 23;126(Pt 10):2304-11. Epub 2003 Jun 23.

Department of Neuroscience, Catholic University, Roma, Italy.

The term seronegative myasthenia gravis (SNMG) refers to the generalized disease without detectable anti-acetylcholine receptor (anti-AChR) antibodies. In these patients, IgG antibodies against the muscle-specific kinase (MuSK) have been described, which reduced agrin-induced AChR clustering in vitro. We have assayed anti-MuSK antibodies in 78 patients with SNMG, who have been followed for many years in our Institution. Here we describe the clinical phenotype of the 37 patients whose results were positive on this assay. MG with anti-MuSK antibodies was characterized by a striking prevalence of female patients (eight men and 29 women). Age of onset ranged from 6 to 68 years, with 56.8% of patients presenting under 40 years of age. All these patients shared a similar pattern of muscle weakness, with prevalent involvement of cranial and bulbar muscles and a high frequency of respiratory crises; the involvement of limb muscles was comparatively less severe and inconsistent. Single-fibre-EMG confirmed the most sensitive examination in the EMG diagnosis of MuSK-positive disease, while, owing to weakness topography, repetitive nerve stimulation in limb muscles was diagnostic in 56.8% of cases. The effect of edrophonium (or neostigmine) injection was equivocal or negative in 11 of 37 patients (29.7%), and the response to oral pyridostigmine was even more unsatisfactory, ranging from mild benefit to overt intolerance. In thymectomized patients, thymus was normal for age or atrophied, and no benefit from surgery was noticed. Thirty-five of 37 patients were given immunosuppressive therapy and 22 received plasma-exchange. The course of the disease was often characterized by periodic exacerbation phases requiring hospitalization and even assisted ventilation; plasma-exchange produced marked improvement in these cases. At the end of the observation period, most patients, although improved, were still symptomatic, having developed permanent facial and pharyngeal weakness together with some atrophy of facial muscles. MuSK-negative disease was comparatively more heterogeneous. Most patients were affected with mild to moderate symptoms and responded well to pharmacological treatment; however, a few subjects in this group had severe refractory disease, poorly responsive to both acetylcholinesterase inhibitors and immunosuppressants.
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http://dx.doi.org/10.1093/brain/awg223DOI Listing
October 2003