Francesca Gulli

Overview of immune abnormalities in lysosomal storage disorders

The critical relevance of the lysosomal compartment for normal cellular function can be proved by numbering the clinical phenotypes that arise in lysosomal storage disorders (LSDs), a group of around 70 different monogenic autosomal or X-linked syndromes, caused by specific lysosomal enzyme deficiencies: all LSDs are characterized by progressive accumulation of heterogeneous biologic materials in the lysosomes of various parts of the body such as viscera, skeleton, skin, heart, and central nervous system. At least a fraction of LSDs has been associated with mixed abnormalities involving the immune system, while some patients with LSDs may result more prone to autoimmune phenomena. A large production of proinflammatory cytokines has been observed in Gaucher and Fabry diseases, and wide different autoantibody production has been also reported in both. Many immune-mediated reactions are crucial to the pathogenesis of different inflammatory signs in mucopolysaccharidoses, and subverted heparan sulphate catabolism might dysregulate cellular homeostasis in the brain of these patients. Furthermore, an inappropriate activation of microglia is implicated in the neurodegenerative foci of Niemann-Pick disease, in which abnormal signalling pathways are activated by impaired sphingolipid metabolism. In addition, not the simple impaired catabolism of gangliosides per se, but also the production of anti-ganglioside autoantibodies contributes to the neurological disease of gangliosidoses. Even if the exact relationship between the modification of lysosomal activities and modulation of the immune system remains obscure, there is emerging evidence of different impaired immunity responses in a variety of LSDs: in this review we investigate and summarize the immune abnormalities and/or clinical data about immune system irregularities which have been described in a subset of LSDs.

Autoimmunity and lymphoproliferation markers in naïve HCV-RNA positive patients without clinical evidences of autoimmune/lymphoproliferative disorders

BACKGROUND HCV can lead to both chronic liver disease and B-cell lymphoproliferative disorders. A strong association exists between HCV and mixed cryoglobulinaemia (MC). METHODS Anti-nuclear antibodies (ANA), rheumatoid factor Ig-G (RF-IgG), free light chain κ and λ (FLC-κ, FLC-λ) levels and κ/λ ratio were evaluated in 50/420 subjects unexpectedly resulted anti-HCV positive after routine screenings for non-hepathological procedures. RESULTS Three/fifty patients had HCV-RNA undetectable in the serum and were excluded from the analysis. Thirty-nine/fifty patients had laboratory evidence of circulating cryoglobulins without liver disease and MC-related symptoms. Among them, 17 resulted ANA-positive. The mean cryocrit was higher in ANA-positive patients, while no other demographic/clinical differences were observed between the groups. Significantly higher levels of RF-IgG were observed in ANA-positive vs ANA-negative patients. κ and λ FLC were higher in ANA-positive patients. A ROC analysis, based on ANA-positivity vs ANA-negativity, confirmed a high sensitivity and specificity of RF-IgG test. CONCLUSIONS Published data concerning MC come mostly from symptomatic vasculitis. We analyzed HCV-patients without MC symptoms, founding cryoglobulins in the majority of them. The increased levels of FR-IgG and FLC in CGs-ANA-positive patients, suggest these test could be used to identify a state of silent autoimmune and/or lymphoproliferative condition before the transition to a frank disease in naïve HCV-patients without symptoms of extrahepatic manifestations.

Assessment of free light chains in HCV-positive patients with mixed cryoglobulinaemia vasculitis undergoing rituximab treatment

Mixed cryoglobulinaemia (MC) is an HCV‐related lymphoproliferative disorder characterized by the presence of circulating immune complexes called cryoglobulins. Treatment with anti‐CD20 monoclonal antibody rituximab is proved to be very useful, especially in patients ineligible to interferon‐based antiviral therapy. Recently, free light chain (FLC) κ/λ ratio and FLC patterns were associated with MC. The aim of this study was to evaluate changes in FLC‐κ, FCL‐λ, FLC ratio following rituximab treatment in patients with HCV‐related MC and to correlate FLC‐κ, FCL‐λ and FLC ratio values with therapy response.

Pre-analytical phase in cryoglobulin (CRG) detection: an alternative method for sample transport.

aEleonora Torti and Maria Teresa Dell’Abate contributed equally to this work. *Corresponding author: Umberto Basile, Department of Laboratory Medicine, School of Medicine, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy, Phone: +39 06 3015 4222, E-mail: umberto.basile@rm.unicatt.it Eleonora Torti and Maria Teresa Dell’Abate: Department of Laboratory Medicine, School of Medicine, Catholic University of the Sacred Heart, Rome, Italy Luigi Colacicco and Cecilia Zuppi: Institute of Biochemistry School of Medicine, Catholic University of the Sacred Heart, Rome, Italy Francesca Gulli and Gian Lodovico Rapaccini: Institute of Internal Medicine, School of Medicine, Catholic University of the Sacred Heart, Rome, Italy Letter to the Editor

Anti-nuclear antibody detection in cryoprecipitates: distinctive patterns in hepatitis C virus-infected patients.

BACKGROUND Anti-nuclear antibodies are immunoglobulins directed against nuclear antigens. They are associated with many autoimmune disorders, but are frequently found in patients infected with hepatitis C virus, possibly indicating an underlying common origin. Likewise, mixed cryoglobulinemia often accompanies autoimmune diseases and hepatitis C infection. AIM To compare anti-nuclear antibodies and immunoglobulin content of cryoprecipitates from hepatitis C virus-positive patients in order to assess their predictive value in the onset of hepatitis C virus-driven extrahepatic disorders. METHODS Serum from 40 hepatitis C virus-positive patients and 50 controls with rheumatoid arthritis was processed for cryoglobulin detection: all subjects presented with Type III mixed cryoglobulinemia. Immunoglobulin content and immunoglobulin subclasses of cryoprecipitates were assessed by immunofixation and tested by ELISA for rheumatoid factor. Cryoprecipitates were also analysed for anti-nuclear antibodies by indirect immuno-fluorescence to identify specific patterns typical of each condition. RESULTS Anti-nuclear antibody patterns differed significantly; 26 infected subjects (65%) were IgG3 positive: of these, 25 were also anti-nuclear antibody-positive (96.1%). CONCLUSIONS IgG3 are autoreactive clones unrelated to viral recognition and possibly involved in autoimmune disorders. Altogether, these results may represent useful diagnostic device for early detection of hepatitis C virus-induced autoimmune diseases.

Relationship between circulating syndecan-1 levels (CD138s) and serum free light chains in monoclonal gammopathies

BackgroundMonoclonal gammopathies encompass a wide range of diseases characterized by the monoclonal expansion of a B-cell clone. Despite emerging therapeutic strategies, chances of survival of patients who are affected are still scarce, which implies that new tools are necessary not only for the diagnosis but also for the follow-up of patients affected by such diseases. In this context, the use of free light chains (FLCs) has been incorporated into many guidelines.Likewise, tumor microenvironment is consistently gaining importance as role player in tumor pathogenesis. Specifically, Syndecan-1 (CD138), a heparan-sulfate proteoglycan is attracting interests as it is highly expressed and shed by myeloma plasma-cells.The aim of our study was to analyze CD138 levels in the serum of patients affected by multiple myeloma or light chain only disease, and to compare the values obtained with free light chain (FLC) kappa, lambda and FLC ratio in both groups of patients.Methods84 patients affected by Multiple Myeloma and Light Chain Myeloma were recruited for this study. Serum CD138 was assessed by ELISA (Diaclone Research, France) and FLC values were quantified by nephelometry (Freelite TM Human Kappa and Lambda Free Kits, The Binding Site, UK). Data was analyzed by GraphPad Prism software and Statgraph.ResultsWe observed higher CD138 mean values in myeloma patients compared to the light chain only myeloma group. A positive linear regression of CD138 and FLC was observed in the light chain only cohort as opposed to myeloma patients which show an inverse trend.ConclusionsThe study highlighted an existing relationship between FLCs and CD138 and wishes to seek also a correlation in order to rapidly and efficiently perform diagnosis and different diagnostic schemes.

Serum free light chain quantitative assays: Dilemma of a biomarker

Serum free light chains detection assays are consistently meeting greater interest for the diagnosis and monitoring of monoclonal gammopathies and plasma cell dyscrasias. Nowadays, there are neither standardized methods nor reference material for the determination of free light chains; for this reason, it is important to compare two different assays used in clinical laboratory.

Heavy chain disease: our experience

Heavy chain disease (HCD) is a rare lymphoproliferative disorder of plasma cells, described for the first time in 1964 by Franklin [1], characterized by production of incomplete monoclonal immunoglobulin heavy chains without associated light chains [2]. HCDs involve the three main immunoglobulin classes, the most common α-HCD, γand μ-HCD. Originally, Franklin’s disease (γ-HCD) was considered to be a lymphoproliferative disease, however, HCDs have variable clinical presentations and different histopathologic features. γ-HCD has been reported to occur equally in men and women, but in a recently reported series there was a clear female predominance [3]. The diagnosis of γ-HCD requires the evidence of a deleted immunoglobulin heavy chain without a bound light chain in the serum or urine [3]. These alterations typically result in loss of a large portion of the constant-1 (CH1) domain of the immunoglobulin heavy chain molecule responsible for light chain binding. In the absence of an associated light chain, the CH1 domain of the normal heavy chain binds to heat-shock protein 78 (hsp78) and undergoes degradation in the proteasome compartment of cells; normal heavy chains unassociated with light chains are therefore never detected in serum or urine. In the HCDs, the altered structure of the CH1 domain prevents the heavy chain from binding both the light chain and hsp78, thereby allowing it to bypass degradation by the proteasome and be secreted into the serum or urine [4]. In addition, the altered heavy chain may function as part of the transmembrane B-cell receptor and facilitate antigen-independent aggregation and downstream signaling by the receptor, thereby conferring a growth advantage to the heavy-chain expressing cells [5]. The clinical course of γ-HCD is extremely variable and ranges from an asymptomatic benign, or stable process to a rapidly progressive neoplasm leading to mortality within a few weeks. Prognosis is variable and the mean survival time has been reported to be 7.4 years (range, 1 month to >21 years) [3, 6]. We describe three cases of γ-HCD associated with chronic lymphocytic leukemia, peripheral T-cell lymphoma, not otherwise specified and small cell lung carcinoma, respectively. All three patients were male. The Ethics Committee of the “Policlinico Universitario A. Gemelli, Roma” approved this study. The first case is a 75-year-old man, who was admitted to the hospital for a 2-months history of progressive worsening of neurological symptoms, i.e. vision changes, dysphagia and amnesia. He had surgery for adenocarcinoma of the rectum 6 years before, with local recurrence after 5 years. In the last hospital stay before admission, he was treated with radiotherapy and chemotherapy with 5-fluorouracil. Total-body computed tomography scanning was negative for local, nodal or other organ involvement. Franklin’s disease was diagnosed 2 years before with no underlying lymphoproliferative or autoimmune disorder. Laboratory tests, including blood leukocyte count and CD4+ to CD8+ cells ratio, were within normal range, but a severe deficiency of serum IgA (0.39 g/L, 0.70–4.00) and IgM (<0.19 g/L, 0.40–2.30) concentrations and a mild IgG deficiency (6.45 g/L, 7.00–16.00) were found. Serum electrophoresis was negative but serum immunofixation electrophoresis (IFE) showed two γ-heavy chain monoclonal components (MCs) migrating in β, not associated with corresponding light chain, and one MC in the γ-region (small MC IgMλ); urine IFE interestingly showed a typical pattern with a broad band of incomplete γ-heavy chain (Figure 1) [7]. *Corresponding author: Umberto Basile, School of Medicine, Department of Laboratory Medicine, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy, E-mail: umberto.basile@policlinicogemelli.it. http://orcid.org/0000-0002-8328-2570 Francesca Gulli, Cecilia Napodano and Krizia Pocino: Department of Laboratory Medicine, Catholic University of the Sacred Heart, Rome, Italy Annarosa Cuccaro and Stefan Hohaus: Institute of Hematology, Catholic University of the Sacred Heart, Rome, Italy

Evaluation of screening method for Bence Jones protein analysis.

*Corresponding author: Umberto Basile, School of Medicine, Department of Laboratory Medicine, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy, E-mail: umberto.basile@policlinicogemelli.it Francesca Gulli, Eleonora Torti, Cecilia Napodano, Maria Teresa Dell’Abate, Elena De Santis and Stefano Angelo Santini: School of Medicine, Department of Laboratory Medicine, Catholic University of the Sacred Heart, Rome, Italy Laura Conti and Giovanni Cigliana: Laboratory of Clinical Pathology, Department of Prevention and Diagnostic Oncology, National Cancer Institute “Regina Elena”, Rome, Italy Cecilia Zuppi: School of Medicine, Institute of Biochemistry and Clinical Biochemistry, Catholic University of the Sacred Heart, Rome, Italy Letter to the Editor

Free light chains of immunoglobulins in patients with systemic sclerosis: correlations with lung involvement and inflammatory milieu

Aim Humoral immunity and B cells are thought to play an important role in the pathophysiology of the systemic sclerosis (SSc). The production of free light chains (FLC) of immunoglobulins is abnormally high in several pathological autoimmune conditions and reflects B cell activation. Furthermore, FLCs demonstrated different biological activities including their capability to modulate the immune system, proteolytic activity and complement cascade activation. The aims of this study are to determine the FLC levels in patients with SSc compared with healthy controls (HC) and to study their possible association with organ involvement and disease characteristics. Methods Sixty-five patients with SSc and 20 HC were studied. Clinical and immunological inflammatory characteristics were assessed for all the patients with SSc. κ-FLC and λ-FLC, interleukin 6 (IL-6) and B cell activating factor levels were measured. Results The mean serum κ-FLC levels and FLC ratio were significantly higher in patients with SSc compared with HC, while the serum λ-FLC levels were comparable. The levels of FLC were comparable in patients with diffuse skin disease and limited skin involvement, while κ-FLC levels were increased in patients with restrictive lung (forced vital capacity (FVC) <80%) disease (26.4±7.4 mg/L) when compared with patients with FVC ≥80% (19.6±7.3 mg/L, P=0.009). In patients with SSc, the levels of serum κ-FLC level directly correlated with the IL-6 levels (R=0.3, P=0.001) and disease activity (R=0.4, P=0.003). Conclusions FLC levels are elevated in SSc and high levels are associated with lung involvement and with a higher degree of inflammation, supporting a possible role of B cell activation in the pathophysiology of the disease.