We hypothesized that progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) reflects the escape of transformed plasma cells from T-cell recognition because of impaired antigen processing-presenting machinery (APM). We studied plasma cells and CD8+ T cells from bone marrow of 20 MGUS patients, 20 MM patients, and 10 control patients. Immunofluorescence and flow cytometry revealed significantly different patterns of APM component expression in plasma cells from the 3 groups. Compared with control patients, MM samples had lower expression of proteasome subunits and peptide transporters and greater expression of chaperones, considering both percentages of stained cells and molecular equivalents of soluble fluorochrome. MGUS samples had intermediate percentages of stained cells but molecular equivalents of soluble fluorochrome similar to control patients. Real-time polymerase chain reaction documented that APM changes occurred at the transcriptional level. Cytotoxicity assays demonstrated that MGUS CD8+ T cells lysed autologous transformed plasma cells more than MM CD8+ T cells did. MGUS progression correlated directly with calnexin, calreticulin, and tapasin and indirectly with delta, LMP2, and LMP10 expression levels; MM disease status did not correlate with APM levels. APM changes may allow transformed plasma cells to elude immunesurveillance in the MGUS-MM pathogenetic sequence.

Autoantibodies to intracellular antigens form a large family of immunoglobulins directed to a variety of ubiquitously expressed intracellular molecules, including numerous enzymes, some ribonucleoproteins and double-stranded DNA. These anti-self antibodies have been found to be selectively expressed in sera of patients with several systemic (non-organ-specific) autoimmune diseases, such as systemic sclerosis (SSc), SLE, mixed connective tissue disease, Sjögren's syndrome and idiopathic myopathies. Despite their important diagnostic and prognostic value and their utility in assessing disease activity, little is known about the molecular mechanisms involved in their generation and role in autoimmune diseases nor is it known why particular autoantibodies are preferentially expressed in certain diseases. Here, we review the different lines of research which are presently being conducted to understand how these autoantibodies are generated (e.g. through apoptotic body formation, molecular mimicry and other mechanisms) and how they encounter antigen in order to cause an autoimmune disease. The recently reported mechanism of intracellular immunity mediated by Ro52 (or tripartite motif containing 21, TRIM21) in a cellular model of adenovirus infection is opening new perspectives for studying the effects of autoantibodies once they get inside cells. © 2011 Elsevier B.V. All rights reserved.

Centromere-associated protein A (CENP-A), a common autoimmune target in a subset of systemic sclerosis patients, appears to have no role to explain why its corresponding auto-antibodies are more frequently found in the limited than the diffuse form of systemic sclerosis. Therefore, we investigated the fine specificity of anti-CENP-A antibodies as a first step to understanding their role in systemic sclerosis pathology. We focused on the amino-terminal portion of CENP-A spanning amino acids 1 to 17 (Ap(1-17)), which represents, along with Ap(17-30), an immunodominant epitope of the protein. Peptide Ap(1-17) was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap(1-17) antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap(17-30). Panning of a phage display peptide library with anti-Ap(1-17) antibodies from 2 patients identified two novel, partially overlapping motifs, <(5)Rx(st)xKP(10)> and <(9)KPxxPxR(15)> as the result of the alignment of specific phage clone insert sequences. Anti-Ap(1-17) IgG from the 8 patients had different reactivities to isolated phage clone insert sequences. Scanning the Swiss-Prot database revealed a large number of different types of proteins containing the two Ap(1-17) antigenic motifs. These data show that anti-CENP-A(1-17) antibodies are generated independently from anti-CENP-B antibodies and display great heterogeneity in their specificity by recognizing different motifs within that peptide sequence. This finding, along with the widespread interspecies and human tissue distribution of the two motifs, suggests that the number of motif-expressing proteins which can be the potential target of these antibodies is markedly higher than that estimated from the peptide-based epitope spreading model.

Background and objective: Systemic sclerosis is a highly invalidating connective tissue disease, whereby an interplay between vasculopathies, fibroblast activation and autoimmunity leads to widespread collagen deposition and tissue fibrosis. Autoimmunity is demonstrated by the presence of anti-nuclear antibodies (ANA) which include different subsets of auto-antibodies (Abs), such as anti-CENP-A Abs. Because CENP-A does not appear to have any pathogenetic (or protective) role to explain why the corresponding auto-Abs are more frequently found in the limited rather than the diffuse form of systemic sclerosis, and to get a deeper insight into the mechanism(s) by which these Abs are generated, we investigated their fine specificity, i.e., the amino acids (motif amino acids) which are recognized by this Ab population. We focused our study on the amino terminal portion of CENP-A spanning amino acid (AA) 1 to 17 (Ap1-17). This region was selected because it represents, along with Ap17-30, an immuno-dominant epitope of CENP-A. Both regions express the motif GPxRx shared with CENP-B, which is thought to be at the basis of anti-CENP-B cross reactivity with CENP-A. Methods: A CENP-A-derived peptide spanning AA 1 to 17 (Ap1-17) was insolubilized on an AffiGel-15 column, which was used for affinity purification of anti-Ap1-17 Abs from anti-CENP-positive sera of 8 patients with systemic sclerosis. SDS-GEL was employed to assess their purity and Western blot to test the reactivity of purified anti-Ap1-17 IgG with recombinant CENP-A and CENP-B. ELISAs were used to test the reactivity of sera or purified anti-peptide Abs. A phage display peptide library (PDPL) was used to identify anti-Ap1-17 Abs-specific motif AA. Web-available programs were looked up for the motif screening against protein database. Results: Anti-Ap1-17 IgG purified from these sera reacted specifically and dose-dependently with Ap1-17, whereas no reactivity was observed with Ap17-30. Panning of PDPL with anti-Ap1-17 IgG from patients pt14 and pt4 identified the 2 partially overlapping motifs &lt;5Rx(st)xKP10.&gt; and &lt;9KPxxPxR15.&gt;. Assessment of the reactivity of isolated phage clones with IgG anti-Ap1-17 divided the 8 patients into 4 groups, in function of their IgG reactivity (or lack of reactivity) with the pt14 and pt4 IgG-specific phage clones (pcs) 14 and pcs4, as follows: group #1 included pt14 IgG reacting with pcs14 only; group#2 included pt4 IgG which reacted with pcs4 only; group #3 included pt5, pt7 and pt9 IgG reacting, though to a different extent, with pc4.22, pc4.26, pc4.33 and pc4.40; group #4 included pt1, pt8 and pt15 IgG reacting neither with Pt4 nor with pt14-specific pcs. It is unlikely that pt1, pt8 and pt15 IgG reacted with the previously defined motif GPxRx expressed also by CENP-B, because of its lack of reactivity with IgG from any of the 8 patients evaluated. Conclusions: Our data show that: a) anti-Ap1-17 are independently expressed from anti-Ap17-30 and from anti-CENP-B; b) two novel motifs can be recognized by anti-CENP-A Abs; and c) IgG anti-Ap1-17 from different patients display a unique specificity, despite the recognition of the same peptide segment.

BACKGROUND: The aim of this study was to identify the main features of a cohort of Caucasian patients with idiopathic (I) and systemic disease-associated (SDA) autoimmune uveitis (AU) who were followed up at a single tertiary reference center. The study consisted of a retrospective analysis of the demographic, clinical, and laboratory features and the response to treatment of 104 patients with AU evaluated between 2004 and 2013, with a median follow-up of 4.8 years. The primary outcome measure was the response to systemic treatment after 24 months of therapy. The data are expressed as the range, percentage, or mean ± standard error. Categorical variables were assessed by Fisher's exact test. RESULTS: The mean age at diagnosis was 40.1 ± 17.8 years for men and 44.1 ± 15.3 years for women. There was a slight female predominance. Of the 104 patients, 72.1% had I-AU and 27.9% SDA-AU. The most frequent associations were with ankylosing spondyloarthritis, autoimmune thyroiditis, inflammatory bowel diseases, and Behcet's disease. Symptoms at presentation consisted of eye redness and pain (28.8%), decreased visual acuity (25.9%), and floaters (18.3%). Complications included cataracts (24%), retinal neovascularization (16.3%), chorio-retinal scars (10.6%), cystoid macular edema (8.6%), glaucoma/ocular hypertension (7.7%), epiretinal membranes (4.8%), and retinal detachment (3.8%). The prevalence of autoantibodies, mostly antinuclear antibodies, was comparable between the I-AU and SDA-AU groups. Fisher's exact test showed a direct correlation between patients with class I HLA B27, Cw8, B5 (51, 52), B51, or Cw2 and the presence of AU, whereas among patients with class II HLA, only DQ1 was a predisposing factor for AU. The therapeutic spectrum included corticosteroids and immunosuppressive agents, given either alone or in various combinations according to the severity of AU and the extent of the clinical response. Among the immunosuppressive drugs, azathioprine was preferentially used for anterior uveitis, and cyclosporine-A for intermediate and posterior uveitis. An assessment of the patients after 24 months of therapy showed a complete remission in 43.3% and a significant clinical improvement in 26.9%. CONCLUSIONS: At our tertiary reference center, the prevalence in Caucasian patients of I-AU was approximately 2.5-fold higher than that of SDA-AU. Our findings point to the need for a patient-tailored therapeutic approach according to the anatomic site and the severity of AU. Therapy should be prolonged, over a period of months and even up to 1-2 years, in order to achieve stable control of the disease and to prevent severe complications. The outcome of SDA-AU is also influenced by treatment of the underlying systemic disease. Additional controlled trials are needed to assess the efficacy and the long-term safety of both the prescribed therapeutic agents and their combinations. KEYWORDS: Ankylosing spondyloarthritis; Autoimmune uveitis; Behcet's disease; Class I and II HLA; Corticosteroids; Immunosuppressive drugs

Autoimmune uveitis (AU), an inflammatory non-infectious process of the vascular layer of the eye, can lead to visual impairment and, in the absence of a timely diagnosis and suitable therapy, can even result in total blindness. The majority of AU cases are idiopathic, whereas fewer than 20 % are associated with systemic diseases. The clinical severity of AU depends on whether the anterior, intermediate, or posterior part of the uvea is involved and may range from almost asymptomatic to rapidly sight-threatening forms. Race, genetic background, and environmental factors can also influence the clinical picture. The pathogenetic mechanism of AU is still poorly defined, given its remarkable heterogeneity and the many discrepancies between experimental and human uveitis. Even so, the onset of AU is thought to be related to an aberrant T cell-mediated immune response, triggered by inflammation and directed against retinal or cross-reactive antigens. B cells may also play a role in uveal antigen presentation and in the subsequent activation of T cells. The management of AU remains a challenge for clinicians, especially because of the paucity of randomized clinical trials that have systematically evaluated the effectiveness of different drugs. In addition to topical treatment, several different therapeutic options are available, although a standardized regimen is thus far lacking. Current guidelines recommend corticosteroids as the first-line therapy for patients with active AU. Immunosuppressive drugs may be subsequently required to treat steroid-resistant AU and for steroid-sparing purposes. The recent introduction of biological agents, such as those targeting tumor necrosis factor-α, is expected to remarkably increase the percentages of responders and to prevent irreversible sight impairment. This paper reviews the clinical features of AU and its crucial pathogenetic targets in relation to the current therapeutic perspectives. Also, the largest clinical trials conducted in the last 12 years for the treatment of AU are summarized and critically discussed.

Belimumab, a specific inhibitor of the soluble B lymphocyte stimulator (BlyS), is the first biological drug approved by the United States Food and Drug Administration for the treatment of patients with active systemic lupus erythematosus (SLE) refractory to standard therapy. Given that an imbalance between regulatory T cells (Treg) and interleukin (IL)-17A-secreting T cells (Th17) has been reported in various autoimmune disorders, we assessed the frequency of both Treg and Th17 peripheral blood populations before and after belimumab administration in 20 patients with active SLE refractory to standard therapy. After six months of treatment, the mean SELENA-SLEDAI score as well as the mean anti-double-stranded DNA antibody titers were significantly decreased. In addition, we observed a significant increase in Treg percentages and a parallel, significant decrease in Th17 percentages, accompanied by significantly reduced serum levels of IL-21. In vitro studies showed that Treg purified from belimumab-treated patients were fully functional and displayed a suppressor function similar to that of Treg purified from healthy donors. Belimumab can restore Treg/Th17 balance in SLE patients with uncontrolled disease activity, and this results in decreased flare rate and reduced glucocorticoid dosage.

INTRODUCTION: In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the 2 immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17. METHODS: Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity. RESULTS: Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA- SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI [greater than or equal to]3 (Fisher exact test, p=0.045) or less restrictive DAI[greater than or equal to]2.5 (p=0.009). CONCLUSIONS: ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lcSSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance.

Cryoglobulins are circulating immunoglobulins that reversibly precipitate at temperatures below 37 °C. Type-II cryoglobulins consist of monoclonal IgM/polyclonal IgG immune complexes (ICs), whereas in type-III cryoglobulins both IgM and IgG are polyclonal. The clinical condition resulting from the presence of cryoglobulins in the blood is called mixed cryoglobulinemia (MC), which can be asymptomatic or manifest as cryoglobulinemic vasculitis (CV). Type-I cryoglobulins, consisting of a single monoclonal isotype, are detected in patients with lymphoproliferative disorders. It is now established that > 90% of MCs are associated with HCV infection. Clinically, the spectrum of symptoms may range in severity from occasional purpuric eruptions to life-threatening features. In addition to the development of liver cirrhosis and hepatocellular carcinoma, the possible progression of HCV-positive CV patients to B-cell non-Hodgkin lymphoma (B-NHL) has been reported. The pathogenetic role played by HCV infection in the onset of B-NHL is suggested by regression of the latter following the achievement of a sustained virologic response (SVR). For several years, interferon-α alone or combined with ribavirin has been the standard of care. However, the rates of clinical, biochemical, and virologic responses have been low, and the occurrence of relapse frequent. The addition of rituximab has resulted in a higher rate of responses. With the advent of direct-acting antiviral agents, SVR has been achieved in ~ 95% of CV patients. However, in a minority of patients, despite SVR, CV may persist or reappear over variable lengths of time from the completion of therapy. The eventual appearance of B-NHL is also possible.

BACKGROUND. Auto-Abs to the histone H3-like centromeric protein-A (CENP-A) are frequently detected in systemic sclerosis (SSc) with limited cutaneous involvement. The long-range goal of this study is to define the origin of CENP-A Abs through the identification of proteins other than CENP-A, which can be their target or induce them. We found that Abs to an immunodominant region of the CENP-A (spanning amino acids 17 to 30; Ap17-30) recognized two distinct, though overlapping, epitopes. One of these is defined by the motif PTPxxGPXXR.OBJECTIVE: 1) to identify proteins expressing the anti-CENP-A-specific motif, 2) to analyze their homology to CENP-A; 3) to screen anti-CENP-A Ab positive sera against peptide-bearing the motif.METHODS: A Swiss-Prot database search was performed to identify proteins expressing anti-CENP-A-specific motifs. Anti-Ap17-30 IgGs were purified from sera by affinity chromatography on an insolubilized peptide column. ELISA was used to test the specificity of serum or Abs against peptides in binding and inhibition assays. Microscopy immunofluorescence assay was performed on fixed-permeabilized HeLa cells, using purified IgG anti-Ap17-30 cells and FITC-conjugated anti-human IgG as detecting reagents. Immunization of BALB/c mice with peptides was performed by intra-peritoneal injections of 10 µg-peptide on day 0, 7, 14 and 70. Sera were drawn weekly until the 13th week and stored at -80°C until used.RESULTS: A database search identified 4 proteins expressing the motif PTPxxGPxxR. Of these, the forkhead box E3 (FOXE3)-derived peptide FOXE3p displayed the highest homology with CENP-A Ap17-30 (identity: 80%), while condensing-2 complex subunit D3-derived peptide (CD3p), protein Jumonji-derived peptide (JMJp) and microtubule-associated protein 1A-derived peptide (MAP1p) displayed 60% homology to Ap17-30. These peptides' antigenic homology with Ap17-30 was evaluated, using IgG anti-Ap17-30 affinity-purified from the sera of 8 anti-CENP-A+ SSc patients (IgG pt1 to pt8). As compared to Ap17-30, Pt1 to pt8 IgG displayed the highest reactivity with FOXE3p, while a lower or no reactivity was observed with the remaining peptides. Furthermore, mouse anti-Ap17-30 and anti-FOXE3 Abs cross-reacted to each other, while Abs elicited with MAP1A partially reacted with CD3. Neither CD3 nor JMJ were found to be immunogenic. Kinetic studies of the reactivity of anti-FOXE3 Abs with Ap17-30 indicated that FOXE3 booster injection on the day 70 markedly increased their relative avidity to Ap17-30. Furthermore mouse anti-FOXE3 sera recognize CENP-A and FOXE3 in western blot, while mouse anti-Ap17-30 recognize CENP-A only. The testing of 69 human anti-CENP-A/Ap17-30+ sera with FOXE3p identified its reactivity with 50.7 % of them.CONCLUSION: 1) FOXE3 displays an antigenic and immunogenic homology with CENP-A-derived Ap17-30; 2) FOXE3p can identify a subset of anti-CENP-A Ab-positive patients. Ongoing studies are expected to establish whether any correlation between FOXE3 reactive Ab-patients and a clinical profile exists.

Background & Aims: Modulation of dendritic cell (DC) function has been theorized as one of the mechanisms used by hepatitis C virus (HCV) to evade the host immune response and cause persistent infection. Methods:We used a range of cell and molecular biology techniques to study DC subsets from uninfected and HCV-infected individuals. Results:We found that patients with persistent HCV infection have lower numbers of circulating myeloid DC and plasmacytoid DC than healthy controls or patients who spontaneously recovered from HCV infection. Nonetheless, DC from patients with persistent HCV infection display normal phagocytic activity, typical expression of the class I and II HLA and co-stimulatory molecules, and conventional cytokine production when stimulated to mature in vitro. In contrast, they do not display the strong switch from immunoproteasome to standard proteasome subunit expression and the upregulation of the transporter-associated proteins following stimulation, which were instead observed in DC from uninfected individuals. This different modulation of components of the HLA class I antigen processing-presenting machinery results in a differential ability to present a CD8+ T cell epitope whose generation is dependent on the LMP7 immunoproteasome subunit. Conclusions: Overall, these findings establish that under conditions of persistent HCV antigenemia, HLA class I antigen processing and presentation are distinctively regulated during DC maturation.

The safety of four different adjuvants was assessed in lupus-prone New Zealand black/New Zealand white (BW)F1 mice. Four groups of mice were injected intraperitoneally with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), squalene (SQU) or aluminium hydroxide (ALU). An additional group received plain phosphate-buffered saline (PBS) (UNT group). Mice were primed at week 9 and boosted every other week up to week 15. Proteinuria became detectable at weeks 17 (IFA group), 24 (CFA group), 28 (SQU and ALU groups) and 32 (UNT group). Different mean values were obtained among the groups from weeks 17 to 21 [week 17: one-way analysis of variance (anova) P = 0·016; weeks 18 and 19: P = 0·048; weeks 20 and 21: P = 0·013] being higher in the IFA group than the others [Tukey's honestly significant difference (HSD) post-test P < 0·05]. No differences in anti-DNA antibody levels were observed among groups. Anti-RNP/Sm antibody developed at week 19 in only one CFA-treated mouse. Mean mouse weight at week 18 was lower in the ALU group than the IFA (Tukey's HSD post-test P = 0·04), CFA (P = 0·01) and SQU (P < 0·0001) groups, while the mean weight in the SQU group was higher than in the IFA (P = 0·009), CFA (P = 0·013) and UNT (P = 0·005) groups. The ALU group weight decreased by almost half between weeks 29 and 31, indicating some toxic effect of ALU in the late post-immunization period. Thus, SQU was the least toxic adjuvant as it did not (i) accelerate proteinuria onset compared to IFA; (ii) induce toxicity compared to ALU or (iii) elicit anti-RNP/Sm autoantibody, as occurred in the CFA group.

Rheumatoid arthritis (RA) is an immune-mediated disease involving chronic low-grade inflammation that may progressively lead to joint destruction, deformity, disability and even death. Despite its predominant osteoarticular and periarticular manifestations, RA is a systemic disease often associated with cutaneous and organ-specific extra-articular manifestations (EAM). Despite the fact that EAM have been studied in numerous RA cohorts, there is no uniformity in their definition or classification. This paper reviews current knowledge about EAM in terms of frequency, clinical aspects and current therapeutic approaches. In an initial attempt at a classification, we separated EAM from RA co-morbidities and from general, constitutional manifestations of systemic inflammation. Moreover, we distinguished EAM into cutaneous and visceral forms, both severe and not severe. In aggregated data from 12 large RA cohorts, patients with EAM, especially the severe forms, were found to have greater co-morbidity and mortality than patients without EAM. Understanding the complexity of EAM and their management remains a challenge for clinicians, especially since the effectiveness of drug therapy on EAM has not been systematically evaluated in randomized clinical trials. Keywords: Rheumatoid arthritis; Extra-articular manifestation; Co-morbidities; Classification; Incidence/rate; Vasculitides; Amyloidosis; Felty's syndrome

This review is based on our experience with ten patients diagnosed with Goodpasture's disease (GD). Six of the patients presented with combined renal and pulmonary insufficiencies; in the remaining four patients the clinical findings were limited to renal involvement. Circulating anti-glomerular basement membrane (GBM) autoantibodies were detected at diagnosis in all patients. Two patients were double-positive for anti-GBM and anti-proteinase-3 neutrophil cytoplasmic antibodies (c-ANCA). Another patient was double positive for anti-GBM and anti-myeloperoxidase cytoplasmic antibodies (p-ANCA). Four patients with rapidly progressive glomerulonephritis underwent hemodialysis: two of these patients died 6 and 8 months after diagnosis, and the other two required maintenance dialysis. The remaining six patients were administered variable combinations of plasma-exchange, corticosteroids, and immunosuppressive drugs, which resulted in a remarkable and progressive improvement in renal function and one-year renal survival in all of them. Building on these observations, we provide an update on this relatively rare, frequently severe, and sometimes lethal autoimmune disease of unknown etiology. GD patients typically present with rapidly progressive renal insufficiency and pulmonary hemorrhage. Involvement restricted to the kidneys alone, as in our series, is also seen. The unfailing immunological hallmark of the disease is the occurrence of circulating anti-GBM antibodies, whose titer is directly related to the clinical severity of GD. The antibodies are associated with serum ANCAs in 10% to almost 40% of GD patients, with double positivity indicative of a worse renal prognosis. The target antigen of anti-GBM antibodies is a component of the non-collagenous-1 (NC1) domain of the α3 chain of type IV collagen, α345NC1. The prevalent expression of this hexamer on the basement membrane of both the glomeruli and the pulmonary alveoli accounts for the frequently combined renal and pulmonary involvement. A strong positive association of GD with the HLA-DRB1*15:01 allele has been described, but the factor(s) responsible for the loss of self-tolerance to NC1 autoantigen has not yet been identified. A conformational change in the quaternary structure of the α345NC1 likely plays a crucial role in triggering an immune response and justifies the proposed description of GD as an autoimmune “conformeropathy.” The function of autoreactive T-cells in GD is poorly defined but may involve a shift from TH2 to TH1 cytokine regulation, such that affinity maturation and the antigen specificity of the antibody response are enhanced. The timely diagnosis of GD and the adoption of a triple therapeutic regimen comprising plasmapheresis, corticosteroids, and immunosuppressive drugs have remarkably improved the previously dismal outcome of these patients, resulting in a one-year survival rate of 70–90%.

To explore the relationship between innate immunity and hepatitis C Virus (HCV) in determining the risk of cirrhosis (CIR), hepatocellular carcinoma (HCC), mixed cryoglobulinemia syndrome (MCS) and non-Hodgkin lymphoma (NHL), we investigated the impact of the toll-like receptor-2 (TLR2) and interleukin-28B (IL28B) genetic variants. TLR2 -174 del variant was associated with TLR2 expression and with specific downstream molecules that drive the expression of different interleukins; rs12979860 Il28B was important in response to interferon-treatment and in spontaneous clearance of HCV. The risk for liver and lymphoproliferative diseases in HCV progression was clarified by stratifying 862 HCV-positive patients into groups based on liver (CIR, HCC) and lymphoproliferative HCV-related diseases (MCS, NHL) and compared with chronic HCV (CHC) infection. Analysis of TLR2-IL28B haplotypes showed an association of wild type haplotype with the lymphoproliferative diseases (OR 1.77, p = 0.029) and a slight increase in HCV viral load (HR 1.38, p = 0.054). Wild type haplotype (TLR2 ins/ins- IL28B C/C) was also found associated with older age in patients with an hepatic diseases (in CIR and in HCC p = 0.038 and p = 0.020, respectively) supporting an effect of innate immunity in the liver disease progression. TLR2 and IL28B polymorphisms in combination showed a role in the control of HCV viral load and different HCV disease progression.

Serial plasma aliquots (50 mL) obtained from 10 commercial donors who converted from hepatitis C virus (HCV) RNA negative to positive were transfused into 2 chimpanzees to assess infectivity during early HCV infection. Plasma, obtained 4 days before HCV RNA detectability by licensed assays, transmitted HCV infection to chimpanzee X355. The infectious PCR-negative plasma was subsequently shown to be positive in 2 of 23 replicates using a sensitive transcription-mediated amplification (TMA) assay, and estimated to contain 1.2 HCV RNA copies/mL (60 copies/50 mL transfused). Plasma units obtained up to 8 weeks earlier were not infectious in a second susceptible chimp, even when from donors with low-level, intermittent HCV RNA detection. Chimp x355 developed acute viremia with subsequent seroconversion, but cleared both virus and Ab in 17 weeks. When rechallenged 38 months later with 6000 RNA copies/mL from the same donor, X355 was transiently reinfected and again rapidly lost all HCV markers. We conclude that: (1) transfusions can transmit HCV infection before RNA detection, but the interval of test-negative infectivity is very brief; (2) early "blips" of HCV RNA appear noninfectious and can be ignored when calculating residual transfusion risk; and (3) markers of HCV infection can be lost rapidly after exposure to low-dose inocula.

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to all CD8+ T-cell adaptive immune responses, including those against tumors. The generation of peptides and their loading on MHC class I molecules is a multistep process involving multiple molecular species that constitute the so-called antigen processing and presenting machinery (APM). The majority of class I peptides begin as proteasome degradation products of cytosolic proteins. Once transported into the endoplasmic reticulum by TAP (transporter associated with antigen processing), peptides are not bound randomly by class I molecules but are chosen by length and sequence, with peptidases editing the raw peptide pool. Aberrations in APM genes and proteins have frequently been observed in human tumors and found to correlate with relevant clinical variables, including tumor grade, tumor stage, disease recurrence, and survival. These findings support the idea that APM defects are immune escape mechanisms that disrupt the tumor cells’ ability to be recognized and killed by tumor antigen–specific cytotoxic CD8+ T cells. Detailed knowledge of APM is crucial for the optimization of T cell–based immunotherapy protocols.

Mucine-1 (MUC1) increases in primary lung disease; however, no data are available on pulmonary arterial hypertension (PAH). Our aim was to analyze MUC1 in PAH and a possible link with pulmonary artery pressure (PAPs), PaO2, PaCO2 and cell-mediated immunity.

Background and objective: In a subset of systemic sclerosis (SSc) patients (pts), anti-CENP-A Ab cross reacts with a FOXE-3-derived peptide (FOXE3p), which presents a striking homology with one (Ap17-30) of the 2 immunodominant epitopes of CENP-A. Preliminary analysis, obtained by absorbance binding, showed that active disease was less likely to occur in anti- FOXE-3+ patients as compared to anti- FOXE-3- patients. Here, we have set up a titration assay using appropriate standards to quantify IgG anti-FOXE-3 and to define whether any clinical correlate exist with the presence of anti FOXE3. As control, clinical correlates with the level of antibodies to Ap17-30 and to the 2nd immunodominant epitope of CENP-A (spanning amino acids 1-to 17, Ap1-17) were also evaluated. Methods: Ap17-30, FOXE3p and Ap1-17 were synthesized at Primm peptide synthesis service (Naples, Italy). The reactivity of serum IgG to specific peptides was measured by indirect ELISAs, using BSA-conjugated peptide as coating reagents and HRP-conjugated xeno-Ab to the Fc portion of human IgG as probe. Sera from 117 SSc pts were tested for anti-centromere and anti-topoisomerase Ab (Diamedix). Calibration curves to measure the levels of Ab were settled, using IgG from sera purified by affinity column coupled to Ap17-30 and Ap1-17 peptides. Clinical records were reviewed to achieve demographic data and information about organ involvement and disease activity, previously evaluated according to a core set of 10 variables. Control sera were obtained from SLE (#47 pts) and from healthy blood donor (HBD) (#25 pts). Results: Purify IgG anti-Ap1-17 and anti-Ap17-30 were used to set-up a standard calibration curve to measure serum levels of anti-Ap1-17 (detection limit range: 2.68-6037ug/ml) and Ap17-30 (0.36-32.85 ug/ml) respectively. As anti-Ap17-30 cross-react with FOXE-3, calibration curve to measure serum levels of Abs to this peptide was achieved using purified anti-Ap17-30 IgG and FOXE-3p as coating reagents (detection limit range: 9.25-880 ug/ml). Of 117 SSc sera, 68 (58.1%) were anti-CENP positive (CENP+) and 94% (#65) of them reacted with Ap17-30 and/or Ap1-17, while no reactivity was observed on the replacement of CENP+ with CENP-, SLE, and HBD sera. The best cut-off concentration of Ab levels able to discriminate CENP+ from CENP- patients (obtained with ROC analysis) were &gt;10.4 ug/ml (sensitivity: 86.8, specificity 98%), &gt;1.2ug/ml (sens. 83%, spec. 96%) and &gt; 74.5 ug/ml (sens.&gt;52.6%, spec. 90%) for anti-Ap1-17, anti-Ap17-30 and anti-FOXE3p respectively. Following dichotomization of CENP+ patients according to their disease activity index score in group 1 (61 patients with score &lt;3), and group 2 (7 patients with score&gt; 3; active disease), a Fisher exact test showed a statistical association of FOXE3p+ sera with group 1 patients (p=0.003, OR: 39.5). Conclusion: Our data confirm the identification of a subset of patients (FOXE3+) that are less likely to have active disease.

Severe pulmonary arterial hypertension (PAH) is rarely observed as the initial manifestation of systemic lupus erythematosus (SLE), and the diagnosis is often delayed. Here we present the case of a 32-year-old woman with severe PAH as the initial manifestation of SLE, who was successfully treated with mycophenolate mofetil and cyclosporine. This case offered the opportunity to critically review the epidemiology data, predictive markers, and pathogenic pathways of SLE-associated PAH (SLE-PAH) in relation to the currently available therapeutic options and to the main clinical trials of the last 10 years focused on the treatment of SLE-PAH. Mycophenolate mofetil and cyclosporine - currently used in the maintenance phase of the disease in certain clinical settings - should be considered, as an alternative to cyclophosphamide, in future clinical trials aimed at evaluating the most effective treatment of SLE-PAH at presentation.

For 50 years Cyclophosphamide was the main drug for Systemic Lupus Erythematosus (SLE) therapy. SLE is a connective tissue disorder that involves many organs. Despite it became a chronic disease, it is still now a killing disease that usually arises in young women. Belimumab is a new drug approved for SLE treatment. It blocks plasma free cytokine BAFF involved in B cell lymphocyte activation. This treatment result as decreasing in disease severity as well as a reduction in steroids usage. We enrolled in Belimumab treatment three patients with a mild to severe disease evaluated with SLE activity index (SLEDAI). All these three patient were non responsive to other treatments and have to take prednisone more than 15 mg per day. Before starting treatment, they referred fatigue and joint pain.No severe renal disease was found. Patients presented a decrease of symptoms, an increase in physical activity with a reduction of disease severity, as confirmed by a reduction of >4 points in SLEDAI score. Prednisone was slowly reduced to 7.5 mg per day or less. No grade 4 toxicities have been found. In the longest follow-up patient hepatic steatosis was revealed.