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Lleixa and Caballero-avila et al. report that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis. Multiple sclerosis is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 or neurofascin-155 have been previously described in multiple sclerosis patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against neurofascin-155, anoctamin-2 and flotillin-1/2 complex in multiple sclerosis. Serum (n = 252) and CSF (n = 50) samples from 282 multiple sclerosis patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-flotillin-1/2, anti-anoctamin-2 and anti-neurofascin-155 antibodies were tested by cell-based assays using transfected cells. We identified six multiple sclerosis patients with antibodies against the flotillin-1/2 complex (2.1%) and one multiple sclerosis patient with antibodies against anoctamin-2 (0.35%). All multiple sclerosis patients were negative for anti-neurofascin-155 antibodies. Three of the anti-flotillin-1/2 positive patients showed anti-flotillin-1/2 positivity in other serum samples extracted at different moments of their disease. Immunoglobulin G subclasses of anti-flotillin-1/2 antibodies were predominantly one and three. We confirm that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis.

The aim of this study is to evaluate the long-term efficacy, safety, and impact on immunoglobulin G (IgG) levels of rituximab in patients with myasthenia gravis (MG). A retrospective, observational study of drug-refractory MG patients treated with rituximab was done. The MG Foundation of America postintervention status (MGFA-PIS) was used to evaluate clinical response. Serum IgG levels were determined at baseline and post-treatment. Hypogammaglobulinemia was defined as IgG7g/L. Thirty patients were included, 12 with anti-MuSK and 18 with anti-AChR antibodies. Mean (SD) follow-up was 85.5 (48) months. All 12 MuSK+ patients but only six (33%) AChR+ patients achieved minimal manifestations or remission (p0.01). Nine severe infections were observed in five patients (17%). One patient was diagnosed with progressive multifocal leukoencephalopathy. At baseline, two patients (2/24; 8%) had hypogammaglobulinemia. During follow-up, hypogammaglobulinemia was observed in 60% (3/5) of patients who developed an infection and in 33% (7/21) who did not. Two of these patients died of infection-related complications. This study supports the effectiveness of rituximab in patients with MG, especially those with anti-MuSK antibodies. Severe infections may appear after rituximab treatment and hypogammaglobulinemia might play a role on it. A standard protocol would be needed to closely monitor IgG levels in MG patients treated with rituximab.

Background and ObjectivesEvidence regarding the safety and efficacy of messenger RNA (mRNA) vaccines in patients with myasthenia gravis (MG) after immunosuppressive therapies is scarce. Our aim is to determine whether the mRNA-1273 vaccine is safe and able to induce humoral and cellular responses in patients with MG.MethodsWe performed an observational, longitudinal, prospective study including 100 patients with MG of a referral center for MG in our country, conducted from April 2021 to November 2021 during the vaccination campaign. The mRNA-1273 vaccine was scheduled for all participants. Blood samples were collected before vaccination and 3 months after a second dose. Clinical changes in MG were measured using the MG activities of daily life score at baseline and 1 week after the first and second doses. A surveillance of all symptoms of coronavirus disease 2019 (COVID-19) was conducted throughout the study. Humoral and cellular immune responses after vaccination were assessed using a spike-antibody ELISA and interferon gamma release assay in plasma. The primary outcomes were clinically significant changes in MG symptoms after vaccination, adverse events (AEs), and seroconversion and T-cell immune response rates.ResultsNinety-nine patients completed the full vaccination schedule, and 98 had 2 blood samples taken. A statistically significant worsening of symptoms was identified after the first and second doses of the mRNA-1273 vaccine, but this was not clinically relevant. Mild AEs occurred in 14 patients after the first dose and in 21 patients after the second dose. Eighty-seven patients developed a humoral response and 72 patients showed a T-cell response after vaccination. A combined therapy with prednisone and other immunosuppressive drugs correlated with a lower seroconversion ratio (OR = 5.97, 95% CI 1.46–24.09, p = 0.015) and a lower T-cell response ratio (OR = 2.83, 95% CI 1.13–7.13, p = 0.024).DiscussionOur findings indicate that the mRNA vaccination against COVID-19 is safe in patients with MG and show no negative impact on the disease course. Patients achieved high humoral and cellular immune response levels.Classification of EvidenceThis study provides Class IV evidence that patients with MG receiving the mRNA-1273 vaccine did not show clinical worsening after vaccination and that most of the patients achieved high cellular or immune response levels.

We investigated whether the difference of antigen tube 2 (TB2) minus antigen tube 1 (TB1) (TB22TB1) of the QuantiFERON-TB gold plus test, which has been postulated as a surrogate for the CD81 T-cell response, could be useful in identifying recent tuberculosis (TB) exposure. We looked at the interferon gamma (IFN-g) responses and differences in TB2 and TB1 tubes for 686 adults with QFT-plus positive test results. These results were compared among groups with high (368 TB contacts), low (229 patients with immune-mediated inflammatory diseases [IMID]), and indeterminate (89 asylum seekers or people from abroad [ASPFA]) risks of recent TB exposure. A TB2-TB1 value.0.6 IU.ml(-1) was deemed to indicate a true difference between tubes. In the whole cohort, 13.6%, 10.9%, and 11.2% of cases had a TB2>TB1 result in the contact, IMID, and ASPFA groups, respectively (P = 0.591). The adjusted odds ratios (aORs) for an association between a TB2-TB1 result of >0.6 IU.ml(-1) and risk of recent exposure versus contacts were 0.71 (95% confidence interval [CI], 0.31 to 1.61) for the IMID group and 0.86 (95% CI, 0.49 to 1.52) for the ASPFA group. In TB contact subgroups, 11.4%, 15.4%, and 17.7% with close, frequent, and sporadic contact had a TB2>TB1 result (P = 0.362). The aORs versus the close subgroup were 1.29 (95% CI, 0.63 to 2.62) for the frequent subgroup and 1.55 (95% CI, 0.67 to 3.60) for the sporadic subgroup. A TB2-TB1 difference of.0.6 IU.ml(-1) was not associated with increased risk of recent TB exposure, which puts into question the clinical potential as a proxy marker for recently acquired TB infection. IMPORTANCE Contact tuberculosis tracing is essential to identify recently infected people, who therefore merit preventive treatment. However, there are no diagnostic tests that can determine whether the infection is a result of a recent exposure or not. It has been suggested that by using the QuantiFERON-TB gold plus, an interferon gamma (IFN-gamma) release assay, a difference in IFN-gamma production between the two antigen tubes (TB2 minus TB1) of.0.6 IU.ml(-1) could serve as a proxy marker for recent infection. In this large multinational study, infected individuals could not be classified according to the risk of recent exposure based on differences in IFN- g in TB1 and TB2 tubes that were higher than 0.6 IU.ml(-1). QuantiFERON-TB gold plus is not able to distinguish between recent and remotely acquired tuberculosis infection, and it should not be used for that purpose in contact tuberculosis tracing. Funding Agencies|Department of Clinical Sciences of the University of Barcelona

Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous plasma cell disorder, with a highly variable clinical course. Immunoparesis, defined by total immunoglobulin measurements, has been shown to be an independent risk factor for progression to symptomatic disease. The heavy/light chain (HLC) assay allows precise measurement of the polyclonal immunoglobulin of the same isotype, enabling the evaluation of isotype-matched immunoparesis (IMI). In this study, we prospectively characterized immunoparesis, as determined by HLC measurements, in 53 SMM patients. Severe IMI was present in 51% of patients, while severe IP of uninvolved isotypes (HLC IP) was present in 39%. Most of the patients with severe HLC IP presented with severe IMI, but not the other way around. Isotype specificity of immune suppression was suggested by lower relative values of isotype-matched HLC pairs, both for IgG and IgA SMM. Severe IMI was associated with other risk factors for progression while patients with severe IMI and severe HLC IP showed an even higher risk profile. Both severe IMI and severe IgM HLC IP showed a significantly shorter time to progression. Finally, gene expression analysis demonstrated differences in the bone marrow microenvironment between patients with IMI and IMI plus HLC IP, with an increased expression of genes associated with cytolytic cells. In conclusion, our data supports isotype specificity of early immunoglobulin suppression mechanisms. While suppression of both involved and uninvolved isotypes is associated with risk of progression, the later appears to develop with more advanced disease and could be mediated by different mechanisms.

Objective: to investigate the feasibility of the New Zealand White (NZW) rabbit to study human acellular dermal matrix (hADM) biomaterials in pelvic reconstructive surgery. Study design: 20 white female NZW rabbits were randomized into 2 groups, the experimental group (hADM graft) and the control group (Polypropylene graft; PP). In each animal, grafts were surgically implanted subcutaneously in the abdominal wall and in the vaginal submucosa layer for 180 days. The graft segments were then removed and the surgical and clinical results were analysed. Results: The main surgical challenges were: a) to obtain the adequate vaginal exposure and the maintenance of the integrity of the vaginal mucosa layer; b) to keep aseptic conditions; c) to locate and dissect the breast vein during abdominal surgery; and, d) to withdraw blood samples from the ear artery. The most abnormal findings during the explant surgery were found in the PP group (33% of vaginal mesh extrusion) in comparison with the hADM group (0% of vaginal graft extrusion), p=0.015. Interestingly, macroscopic observation indicated that the integration of the vaginal grafts was more common in the hADM group (40%) than in the PP group, in which the vaginal mesh was identified in 100% of the animals (p=0.014). Conclusions: The NZW rabbit is an excellent model to assess materials to be used as grafts for pelvic reconstructive surgery and vaginal surgery. Animals are easily managed during the procedures, including the surgical intervention and vaginal mucosa handling.

BACKGROUND Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous clonal plasma cell disorder. Prognostic factors to identify patients at higher risk of progression have been described, including the suppression of uninvolved polyclonal immunoglobulins (Ig) or immunoparesis (IP). Novel serum assays allow the quantification of each isotype-specific heavy and light chain pair (HLC), enabling the study of a new type of IP: the suppression of the uninvolved light chain counter part of the same heavy-chain Ig isotype (isotype matched immunoparesis). AIM: To prospectively characterize the prevalence and severity of immunoparesis as determined by HLC measurements, its association with other risk factors for progression and its prognostic significance, in patients with SMM diagnosed at a single institution. METHODS: Fifty-four patients newly diagnosed with SMM from January 2014 through March 2019 were prospectively included. Serum samples obtained at baseline and during follow-up were tested for M-protein level, free light chain (FLC) and HLC concentrations (all three isotype pairs at baseline, involved isotype pair at follow up visits). Isotype matched immunoparesis (IMI) was defined by the HLC assay, based on the levels of the Ig with the same heavy chain of the monoclonal isotype but of the alternative light chain (e.g. IgG-kappa suppression in a patient with IgG-lambda SMM). Aditionally, HLC immunoparesis (IP) was defined by suppression of any Ig with a heavy chain different to the M-protein (e.g. IgA kappa/lambda pair suppression in a patient with IgG-lambda SMM). Severe immunoparesis was defined by Ig values suppressed by 50% or greater below the lower limit of normal (LLN). RESULTS: Median age at diagnosis was 73 years, with the following heavy chain isotype distribution: 31 IgG, 20 IgA, 2 biclonal IgG-IgA and 1 light-chain only SMM. Median follow up for the series was 2.5 years. Isotype specificity of immunoparesis was reflected in the greater prevalence of IMI vs. noninvolved isotype IP (Figure 1, A). HLC measurements identified IMI in 42 out of the 53 patients with IgG, IgA or biclonal SMM (82%), with severe IMI observed in 27 patients (53%). Suppression of at least one HLC pair of an uninvolved isotype was present in 37 patients (72%), with severe suppression observed in 20 patients (38%). Eleven patients presented severe IMI without severe IP of uninvolved isotypes. On the other hand, only one out of the 37 patients with IP of uninvolved isotypes presented without IMI. In the case of the 12 patients that showed IP of any IgM HLC pair, they all had concomitant severe IMI and IP of the other uninvolved heavy chain isotype. Analysis of other risk factors for progression between groups of patients with i) no severe immunoparesis (n=22), ii) severe IMI without severe IP of uninvolved isotypes (n=11) and iii) severe IMI and severe IP of one or more HLC pairs of uninvolved isotypes (n=18), showed significantly different FLC ratios and lower % of normal plasma cells in bone marrow (Figure 1, B). There was a trend, although statistically not significant, towards higher M-protein levels. Progression to malignant symptomatic gammopathy was observed in 12 patients. The suppression of any IgM HLC pair was associated with a shorter time to progression (p=0.007; HR=4.2; 95% CI, 0.84-21) (Figure 1, C). Severe IMI alone or severe IMI plus severe IP of a different isotype were associated only with a statistically not significant trend towards a shorter time to progression. Eight patients demonstrated an evolving behavior (≥ 10% increase in the involved HLC pair in 3 or more consecutive determinations from diagnosis), 7 of which presented with severe IMI at diagnosis. Of the five evolving patients that progressed, four had severe IMI and the remaining one developed severe IMI during follow up. Patients with severe IMI at diagnosis maintained the same level of HLC suppression throughout the follow up period. CONCLUSION: The significantly greater incidence of IMI over IP of uninvolved isotypes pairs supports an isotype specificity of early Ig suppression mechanisms in the case of IgG and IgA SMM. That would be of special interest at the time of initial evaluation of patients with SMM using HLC pairs. Both IMI and IP of uninvolved isotypes are associated with other recognized risk factors for progression, but the later appears to develop with more advanced disease and associate with a higher risk of progression. Disclosures Cibeira: Amgen: Honoraria, Other: Educational lectures; Celgene: Honoraria, Other: Educational lectures; Akcea Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational lectures. Bladé:Oncopeptides: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria.

Background: The antibody against the transcriptional intermediate factor 1γ (Anti-TIF1γ or anti-p155), is considered specific for dermatomyositis (DM) and its a marker of cancer risk. Our center carries out the determination of anti-TIF1γ by a homemade immunoblot (IB) technique with a 155 kDa TIF1γ human recombinant protein (OriGene). Currently there is a commercial technique of IB to detect TIF1γ (Euroimmun), however, commercial kits sometimes elaborate antigens without using whole protein structure. To date, there are no studies that study the agreement between the commercial kit and homemades IB. Objectives: 1-To compare the commercial IB results of anti-TIF1γ with homemade IB. 2-To describe and compare the clinical characteristics of the patients analyzed by both techniques. METHODS: Observational retrospective study that included adult patients with some determination of anti-TIF1γ antibodies since 2014 in two tertiary-level university hospitals. We collected demographic and clinical data of all patients. Taking into account their diagnoses we grouped them into: Cancer associated Myositis (CAM), DM without cancer, Systemic autoimmune Diseases (SAD) non-DM and other diagnoses. Subsequently, the clinical agreement with the IB results of anti-TIF1γ performed by commercial (cIB) and homemade (hIB) technique were analyzed. A p value RESULTS: 48 patients were recruited (77.1% women). 13 had diagnosis of CAM, 19 of DM without cancer, 7 of other non-DM EAS, and 9 had other diagnoses. Of all DM (CAM and DM without cancer), 12 were positive for both techniques. 5 presented discordant results (4 hIB positive with negative cIB and 1 cIB positive with negative hIB). 15 DM were negative for the two techniques, of which 8/15 had other antibodies associated with autoimmune Myopathy (3 NXP-2, 1 Mi2, 1 MDA5, 1 SAE, 1 Jo1 and 1 Ro52). When we analyzed patients with CAM with both techniques, 10/13 presented positive hIB vs 8/13 cIB positive. DM without cancer, 6/19 had positive hIB vs 5/19 cIB positive. The sensitivity and specificity of each test was evaluated in CAM and DM without cancer, being 61.5% and 73.7% respectively for cIB (p = 0.071), compared to 76.9% and 68.4% for hIB (p = 0.029). In non-DM SAD group, 1 patient had positive determination for both techniques, 3 had positive cIB with negative hIB, and 3 cIB negative with positive hIB. We also found other antibodies in 6/7 patients with non-DM SAD. The 9 patients with other diagnoses were: 1 positive for both techniques and 8 positive for cIB and negative for hIB. CONCLUSIONS: Homemade IB with 155 kDa recombinant protein was superior to cIB to detect TIFγ in our serie of patients with CAM. This could have clinical implications in screening and early detection of neoplasia that affect false negative by cIB. A positive cIB in patients not suspected as a SAD were negative for hIB in the major part of cases. It would be recommended to check the commercial IB using homemade IB, due to its known clinical-therapeutic implications. Disclosure of interests: Ana Milena Millan arciniegas: None declared, M. ANGELES MARTINEZ: None declared, aNAIS MARISCAL: None declared, aNDRES BAUCELLS: None declared, Leticia alserawan: None declared, Ernesto Trallero: None declared, Cesar Diaz-Torne: None declared, ana Laiz Consultant for: Lilly, Novartis, abbVvie, MSD, UCB and Janssen, Speakers bureau: Lilly, Novartis, abvvie, MSD, UCB and Janssen, Berta Magallares: None declared, Patricia Moya: None declared, HyeSang Park: None declared, andrea Garcia-Guillen: None declared, Sycille Jeria: None declared, DAVID LOBO: None declared, albert Gil: None declared, Coral Gozalvez: None declared, Susana P. Fernandez-Sanchez: None declared, Laura Martinez-Martinez: None declared, Esther Moga: None declared, Hector Corominas: None declared, Candido Juarez: None declared, Ivan Castellvi Consultant for: I received fees less than 5000USD as a consultant for Kern and actelion, Paid instructor for: I received fees less than 2000USD as a instructor for Boehringer -Ingelheim, Novartis and Gebro, Speakers bureau: ND, albert Selva-O’Callaghan: None declared

Psoriasis is currently considered to be an immune-mediated disease whose patho-mechanisms involve platelet activation, which seems to correlate with the activity of the disease. Platelet activation is associated with the formation of platelet-lymphocyte complexes (PLyC), although their significance remains unknown. Moreover, biological treatments that target tumor necrosis factor-α (TNF-α) reduce platelet activation. To clarify the significance of PLyC, we compared their levels in patients with psoriasis with those of healthy donors and determined whether platelet binding modifies the secretion of IL-17A by T helper cells. Finally, we assessed the effect of anti–TNF-α treatment on PLyC in responder and non-responder patients with psoriasis. Ours results demonstrated an increase in PLyC in patients with psoriasis. Moreover, the percentage of IL-17-secreting cells was observed to be higher in the platelet-lymphocyte complex population, and these cells tended to secrete greater amounts of IL-17A. Psoriasis patients treated with anti–TNF-α normalized platelet-lymphocyte complex values, and the basal percentage of platelet-T helper lymphocyte complexes was significantly higher in the responder group. In conclusion, PLyC are increased in psoriasis patients, and the number of complexes decreases in response to anti–TNF-α treatment, specifically in the responder group of patients. This finding suggests that PLyC are a prognostic biomarker of response to anti–TNF-α therapy, but prospective studies are necessary to verify these results in patients with psoriasis.

Background:Cryoglobulinemia (CG) is a rare phenomenon, which is defined as the persistent presence in serum of abnormal immunoglobulins (Igs) that precipitate in vitro at less than 37°C and dissolve when the temperature rises again. Is related to hematological disorders, infections and autoimmunes diseases.Objectives:To describe the differential clinical features, serological and demographics in a cohort of patients diagnosed with CG.Methods:We describe a retrospective cohort of 252 cryoglobulin (Cg) positive samples, obtained from a database from the immunology laboratory of a tertiary hospital (November 2018-November 2019). We obtained 182 patients with CG, classified according to their etiology into 4 groups: 1)Rheumatic diseases (RD) that includes rheumatoid arthritis, Systemic lupus erythematosus, Sjögren´s syndrome and Systemic scleroderma, 2)Hepatotropic viruses (HV) with patients diagnosed with Hepatitis C virus, B virus and both, 3)Hematological diseases (HD) and 4)Essential cryoglobulinemia (CGE). Demographic variables, clinical and serological data were collected. A comparative analysis was performed with the Mann-Whitney U test and the multivariate Kruskal-Wallis test, nonparametric variables were compared using a Wilcoxon test. Ten patients, with more than one disease from 4 groups, were excluded from the study.Results:Out of 182 reviewed patients, 172 were included in the study. Mean age at diagnosis was 59.7(±14.0). Demographic, clinical and laboratory characteristics are described in table 1. Mixed CG was the predominant subtype, in 116 (67.4%) patients. The most prevalent CG-associated diseases were HV infection with 91(53%) patients. CGE mostly presented with cutaneous manifestations (p=0.0001), particularly purpura. In RD group the presence of Raynaud and non-erosive arthritis (p=0.0001) was relevant. Laboratory findings showed that CG titration varies according to the etiology, being HD the one with the highest values with 292.2 (±546.2). There is significant difference in terms of the average of rheumatoid factor (RF) being higher in the group by CGE. On other hand, the group HV presented more consumption of complement, and showed the lowest average p=0.0001, without more severe clinical manifestations.RD (n=47)HV (n=91)HD (n=17)CGE (n=17)Gender,n(%) F42 (89.4)57 (62.6)7 (41.2)11 (64.7)Age at dg, years, (± SD)60.6 (±14)59.6 (±13.1)61.1(±16.6)56.3(±20.8)p=0.8CLINICAL CHARACTERISTICSSkin n (%)18 (38.3)10 (11.0)2 (11.8)9 (52.9)pRaynaud n (%)14 (29.8)1 (5.9)3 (17.6)pPurpura n (%)6 (12.8)9 (9.9)2 (11.8)6 (35.3)p=0.04Acrocyanosis n (%)6 (12.8)1 (5.9)p=0.0033Ulcers n (%)3 (6.4)2 (2.2) -2 (11.8)p=0.19Peripheric Neuro n (%)10 (21.3)9 (9.9)1 (5.9)4 (23.5)p=0.13N-E arthritis n (%)22 (46.8)8 (8.8)1 (5.9)4 (23.5)pGMN n (%)5 (10.6)3 (3.3)1 (5.9)3 (17.6)p=0.11LABORATORYCg (mg/dL) x (± SD)26.7 (±63.2)65.8 (±256.5)292.4 (±546.2)47.59 (±79.1)pIsotype IgG, n (%)G+M 26 (55.3)G+M 72 (79.1)M 8 (47.1)G+M 12 (70.6)β2M (≥1.8 mg/L), n (%)7/40 (17.5%)1/5 (20.0%)3/12 (25.0%)-p= 0.44RCP (mg/L) p 5010.3 (±26.2)3.9 (±3.0)13.4 (±18.3)8.5 (±12.0)p= 0.47ESR (mm/h) p5040.0 (±28.5)20.3 (±20.2)35.4 (±35.1)24.5 (±25.0)p= 0.0003RF + (>20UI/mL), n (%)19/46 (41.3)44/86 (51.2)5/11 (45.5)7/17 (41.2)p= 0.09p5090.6 (±175.9)161.0 (±219.5)94.8 (±135.6)284.5 (±619.3)pC3 ()20 (42.6)47 (51.6)3 (17.6)3 (17.6)p= 0.13x (± SD)90.1 (±28.6)68.5 (±10.8)99.1 (±29.0)114.8 (±12.7)pC4 ()17 (36.2)36 (39.6) -3 (17.6)p= 0.02x (± SD)15.6 (±9.0)7.6 (±3.5)20.4 (±7.4)21.1 (±9.5)pConclusion:In our cohort, not all patients with CG presented clinical manifestations being those associated with CGE and RD those with the highest skin and joint expression. The most prevalent association of CG continues to be the HV and we confirmed the characteristic decrease in C3 and C4 complement levels, together with the positivity for RF.Disclosure of Interests:None declared

Background and objectives: Percentages of blood CD19 + CD5 + B cells and CD8 + perforin + T lymphocytes can predict response to Interferon (IFN)-beta treatment in relapsing-remitting multiple sclerosis (RRMS) patients. We aimed to standardize their detection in a multicenter study, prior to their implementation in clinical practice. Methods: Fourteen hospitals participated in the study. A reference centre was established for comparison studies. Peripheral blood cells of 105 untreated RRMS patients were studied. Every sample was analyzed in duplicate in the participating centre and in the reference one by flow cytometry. When needed, participating centres corrected fluorescence compensations and negative cut-off position following reference centre suggestions. Concordance between results obtained by participating centres and by reference one was evaluated by intraclass correlation coefficients (ICC) and Spearman correlation test. Centre performance was measured by using z-scores values. Results: After results review and corrective actions implementation, overall ICC was 0.86 (CI: 0.81-0.91) for CD19 + CD5 + B cell and 0.89 (CI: 0.85-0.93) for CD8 + perforin + T cell quantification; Spearman r was 0.92 (0.89-0.95; p < 0.0001) and 0.92 (0.88-0.95; p < 0.0001) respectively. All centres obtained z-scores 5 0.5 for both biomarkers. Conclusion: Homogenous percentages of CD19 + CD5 + B cells and CD8 perforin + T lymphocytes can be obtained if suitable compensation values and negative cut-off are pre-established.

The aim of this study is to assess the variations of the RF and ACCP in RA patients treated with biologics in actual clinical practice (real) conditions for a one-year follow-up from the first biologic medication. The evaluated patients with a diagnosis of RA, according to the American College of Rheumatology (ACR) 1987 were selected from the outpatient consult of Rheumatology of the “Hospital de Sant Pau” during one month (November 2012). We collected and analyzed data from 41 patients with RA and positivity for RF and/or ACCP. Of the 41 patients had given FR and ACCP at 3, 6 and 12 months respectively in 18 and 10 patients. In 22 patients had given DAS 28 at 3, 6 and 12 months respectively. The mean age of the sample is 55 years (range 29-79), with a mean disease progression 9 years (4 months to 32 years). 70% are women. 33 patients (80.5%) initiated treatment with anti-TNF and 8 (19.5%) with other no anti-TNF mechanism of action. There was a statistically significant (p = 0.001, ANOVA) decrease in DAS 28 (average decrease of 1.6 points) at 3 months is maintained at 6 and 12 m and no significant differences in their evolution by separating anti-TNF drugs vs. other biological agents (different mechanisms of action (p = 0.285). So we have not detected a correlation between DAS 28 and FR or ACCP along the first 12 months of biological treatment. In our experience we did not find a correlation between DAS 28 and RF or ACCP, thus RF and ACCP do not appear to predict the response to treatment.

Introduction: Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous clonal plasma cell disorder. A number of prognostic factors to identify patients at a higher risk of progression have been described, such as the size of the M protein, proportion of abnormal bone marrow plasma cells (BMPCs), immunoparesis and serum free light chain (FLC) k/l ratio. More recently, isotype-specific uninvolved heavy and light chain (HLC) pair suppression measured with the Hevylite assay was also associated with an increased risk of progression. Recent studies have evaluated the key prognostic impact of an increase in M-protein levels during follow-up ("evolving" pattern). However, an important limitation could be the evaluation of M-protein level variations based on serum protein electrophoresis (SPE) in patients with a small size M-spike. The aim of this study was to prospectively analyze the changes in M-protein according to SPE and HLC measurements, as well as other risk factors for progression, in patients with SMM. Methods: Thirty patients newly diagnosed with SMM at a single institution from January 2014 through September 2017 were prospectively included in the study. For each patient, baseline levels of known prognostic factors (serum M-protein, serum and urine immunofixation, clonal BMPCs percentage, total immunoglobulins, involved/uninvolved FLC and involved/uninvolved HLC pairs) were recorded. During the follow up, M-protein level, FLC and isotype specific HLC pairs were also analyzed. Evolving change in M-protein level according to SPE was defined as ³ 10% increase within the first 6 months of diagnosis (if M-protein was ³ 30 g/L) and/or ³ 25% increase within the first 12 months (for any level of M-protein); evolving change according to HLC was defined as a ³ 10% increase in the involved pair. A sequential increase in each of three or more consecutive measurements from diagnosis was considered an evolving change regardless of its magnitude. Results: The clinical characteristics of the total of patients, as well as of the patients with evolving changes in M-protein according to HLC are summarized in Table 1. During the study period, 5/30 (17%) of patients demonstrated an evolving behavior of the M-protein according to SPE. Four of these patients (4/5) also showed a progressive increase in the M-protein in the HLC measurements. One patient showed stable HLC levels even though both the M-protein and the involved FLC progressively increased. This patient was of intermediate and low risk according to Mayo Clinic and PETHEMA scores, respectively. On follow up, no progressive suppression of the isotype-specific uninvolved HLC pair or increase in the FLC ratio was noted, and there have been no signs of progression after a follow up of 3 years. According to involved HLC-pair levels, 12/30 (40%) of patients demonstrated an evolving behavior. Five out of 7 patients that were not classified as evolving by SPE, were IgA isotype. Eight out of 12 patients showed severe isotype-specific suppression of the uninvolved HLC-pair (> 50% below lower level of normal) as well as a highly abnormal FLC ratio (8). Three out of the 4 remaining patients showed either severe isotype-specific HLC pair suppression or highly abnormal FLC ratio in follow up measurements. Compared to patients with no "HLC-evolving pattern", evolving patients were more likely to have highly abnormal FLC ratios (90 vs. 33%, p=0.009), severe suppression of the other isotypes (64 vs. 19%, p=0,024), highly abnormal isotype-specific HLC ratios (67 vs. 33%, p=NS), severe isotype-specific HLC-pair suppression (75 vs. 50%, p=NS), and immunoparesis (67 vs. 39%p=NS). Five patients progressed to symptomatic multiple myeloma during follow up; 4 of them showed a progressive increase in the involved HLC pair from diagnosis. The remaining patient demonstrated a progressive increase in the involved HLC pair that started 19 months prior to progression, followed 4 months later with an increase in M-protein as measured by SPE. Conclusions: In our series, the Hevylite assay allowed us to identify patients with a progressive increase in M-protein (clonal heavy/light chain pair) that was not evident with SPE measurements. This "HLC evolving pattern" was associated with other risk factors for progression to symptomatic disease and with worsening of other prognostic parameters during follow up. Disclosures Rosinol: Janssen, Celgene, Amgen, Takeda: Honoraria. Bladé:Janssen: Honoraria.

Fusion of dendritic cells and tumor cells (FCs) constitutes a promising tool for generating an antitumor response because of their capacity to present tumor antigens and provide appropriate costimulatory signals. CD40-CD40L interaction has an important role in the maturation and survival of dendritic cells and provides critical help for T-cell priming. In this study, we sought to improve the effectiveness of FC vaccines in a murine model of B-cell lymphoma by engineering FCs to express CD40L by means of an adenovirus encoding CD40L (Adv-CD40L). Before transduction with Adv-CD40L, no CD40L expression was detected in FCs, DCs or tumor cells. The surface expression of CD40L in FC transduced with Adv-CD40L (FC-CD40L) ranged between 50 and 60%. FC-CD40L showed an enhanced expression of CD80, CD86, CD54 and MHC class II molecules and elicited a strong in vitro immune response in a syngeneic mixed lymphocyte reaction. Furthermore, FC-CD40L showed enhanced migration to secondary lymphoid organs. Splenocytes from mice treated with FC-CD40L had a dramatic increase in the production of IL-17, IL-6 and IFN-gamma, compared with controls. Treatment with the FC-CD40L vaccine induced regression of established tumors and increased survival. Our data demonstrate that FC transduced with Adv-CD40L enhances the antitumor effect of FC vaccines in a murine lymphoma model and this is associated with an increased Th17-type immune response. Gene Therapy (2010) 17, 469-477; doi: 10.1038/gt.2009.150; published online 10 December 2009

Background: Pathogen infection is a complex process in which several pathogen-recognition receptor (PRR) pathways are activated to induce proinflammatory mediators. The activation of multiple PRRs suggests an interaction between Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptor (NOD) signaling pathways. Methods: To understand the modulation induced by NOD2 signals on successive responses to pathogen-associated molecular patterns (PAMPs), we examined how muramyl dipeptide (MDP) pretreatment reprograms the MDP+LPS (lipopolysaccharide) response of monocytes from human peripheral blood. Results: Preexposure to bacterial MDP components induced selective tolerance to a subsequent NOD2+TLR4 stimulation. MDP pretreatment inhibited the production of tumor necrosis factor alpha (TNFα) and interleuken 10 (IL10), whereas IL6 and IL8 remained unaffected. MDP-induced tolerance was independent of receptor downregulation but was associated with reduced levels of phosphorylated TAK1 and abrogated phosphorylation of the downstream MAPK. Since Nod2 mutations have been associated with susceptibility to develop Crohn's disease (CD), we compared the MDP-induced tolerance in healthy donors and CD patients with compound heterozygous Nod2 mutations (Mut-Nod2) expressing variant NOD2 proteins. MDP-induced tolerance in Mut-Nod2 patients reduced IL10 but not TNFα production. In contrast with healthy donors, a p38-independent TNFα production was observed during the kinetics of the MDP+LPS response in Mut-Nod2 patients. Conclusions: Our findings suggest that the selective tolerance induced by MDP in healthy donors was related to the modulation of a convergent nub of NOD2 and TLR4 signaling pathways. This MDP-induced tolerance was impaired in Mut-Nod2 CD patients, resulting in a p38-independent TNFα production and an imbalance between pro- and antiinflammatory cytokines that could be partly responsible for the pathogenesis of CD. Inflamm Bowel Dis 2009

AIMS In T cells, protein kinase C (PKC) theta plays a major role in T-cell receptor (TCR)-mediated activation of a novel nuclear factor (NF)-kappaB pathway that involves phosphorylation of p65 at serine 536 (Pp65(Ser536)). Bcl-10 acts along the same pathway downstream of PKC theta to activate NF-kappaB. The aim was to investigate the relationship between the expression of PKC theta, Bcl-10 and P-p65(Ser536) proteins and their prognostic significance in peripheral T-cell lymphomas (PTCLs). METHODS AND RESULTS Paraffin-embedded tissues from 30 patients with PTCLs treated with curative intention were evaluated retrospectively. Expression of PKC theta, Bcl-10 and P-p65(Ser536) proteins was assessed using immunohistochemistry. Expression of PKC theta was detected in 22 of 30 cases (73%), Bcl-10 in 20 of 30 (67%) and P-p65(Ser536) in 21 of 30 (70%). Bcl-10+ tumours were associated with PKC theta (18 of 22) (P < 0.0001) and Pp65Ser536 (19 of 21) expression (P < 0.0001). Patients with Bcl-10+ or P-p65(Ser536+) tumours fared better, with a 5-year overall survival of 48 and 45%, respectively, versus 0% for negative tumours (P = 0.029 and P = 0.04, respectively). CONCLUSIONS Bcl-10 is expressed in PTCLs, correlates with PKC theta and Pp65(Ser536) expression and seems to be associated with better survival.

The DRB1* polymorphism in 941 randomly selected individuals from the Umbilical Cord Blood Bank of Barcelona (92.75% of Spanish origin) was determined by sequence-based typing. The HLA profile was similar to that of other Mediterranean populations, with DRB1*0701 and *0301 being the most frequent alleles. This may be a consequence of the mixture of alleles as a result of migration from contiguous geographical areas.

Perivascular adipose tissue has emerged as a critical regulator of vascular function implicated in the pathophysiology of atherosclerosis. Virtually all arteries are surrounded by a significant amount of perivascular adipose tissue. This tissue is a very active endocrine and paracrine source of inflammatory cytokines and adipokines. Moreover, adipocytes express toll-like receptors (TLRs) to be able to respond to lipids and other self and nonself molecules activating proinflammatory pathways. We analyze the TLR/JAK-STAT/SOCS transduction pathways in adipose tissue from patients with atherosclerosis to unravel the mechanisms implicated in the pathophysiology of atherosclerosis.

Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism in the clinical activity of rituximab for treatment of B-cell malignancies. Natural killer (NK) cells, through the activating receptor FcgammaRIIIa (CD16), play a major role in rituximab-mediated ADCC. We have studied the in vitro effect of NK stimulators, such as interleukin-15 (IL-15) and CpG oligodeoxynucleotides A-Class (CpG ODN A), in the enhancement of rituximab-mediated ADCC against B-cell lymphoma.Peripheral blood mononuclear cells (PBMC), purified NK cells, or NK-depleted PBMC from healthy donors, were activated with IL-15 or CpG ODN A, and cocultured with B-lymphoma cells in the presence of rituximab to evaluate the enhancement of the cytotoxicity.The rituximab-mediated ADCC of IL-15-activated PBMC was twofold compared to unstimulated PBMC (73% +/- 7% vs 37% +/- 5% respectively, p0.001). Similarly, rituximab-mediated ADCC was enhanced when PBMC were activated with CpG ODN A as compared to CpG ODN control (61% +/- 11% vs 36% +/- 8%, respectively, p = 0.02). Nevertheless, the ADCC of purified NK cells was increased only with IL-15. NK-depleted PBMC activated with either IL-15 or CpG ODN A showed no ADCC, suggesting that NK are the major effector cells. Furthermore, IL-15 or CpG ODN A-activated PBMC, but not activated purified NK cells, secreted large amounts of interferon-gamma in the presence of rituximab-coated lymphoma cells.IL-15 and CpG ODN A enhance rituximab-mediated ADCC against B-cell lymphoma. Under these conditions, NK cells seem to be the main effector cells mediating ADCC. These findings suggest that these agents could be used as adjuvants in combination with rituximab for patients with B-cell lymphoma.

We report a novel HLA-B*41 allele (HLA-B*4106) initially detected by an unusual sequence-specific oligonucleotide (SSO) hybridization pattern and identified by sequence-based HLA typing. Molecular cloning and sequencing determined that the new HLA-B allele was identical to the HLA-B*4101 in exon 2 and 3 except for a single nucleotide substitution in the exon 3 changing codon 204 from Glu to Gln (GAG-->CAG). In addition, intron 2 was identical to the published HLA-B*4104 intron 2, except for the base 509 (C-->G).

Dendritic cells (DCs) are the main antigen presenting cells and play a pivotal role in the stimulation of T-cell immune responses. DCs cultured in the presence of a single tumor antigen can elicit an immune response against tumor cells expressing that antigen. However, simultaneous use of several tumor antigens may be advantageous since polyclonal activation of T cells against different tumor antigens may be a better approach to eradicate tumor cells. In this sense, fusions of dendritic and tumor cells (FCs) show a broad spectrum of tumor antigens, both known and unidentified, to be presented by class I and II MHC. Although prophylactic vaccines were successful in murine models, the results in the therapeutic setting have been unsatisfactory. We hypothesised that enhancing costimulation of FCs would help to break tumor tolerance once the tumor is established. To this purpose, we transduced FCs with a recombinant adenovirus encoding CD40L (AdvCD40L or AdvGFP as control) and we studied the therapeutic antitumoral effect of the administration of FC-CD40L in a murine model of myeloma. DCs obtained from day 7-bone marrow cultures of Balb/c mice were fused with tumor cells, a syngeneic murine myeloma cell line (4TOO). FCs hybrids were generated with PEG and selected after culturing in HAT medium plus GM-CSF for 7 days. FC were quantified by determining the percentage of cells that coexpress specific DC (CD11c) and tumor markers (CD138). Mean fusion efficiency was 30% (20–40%) and FCs expressed moderate levels of CD80, CD83, CD86, CD54, CD40 and MHC II and did not express CD40L. FC-CD40L showed a significant increase of expression of costimulatory molecules (CD80, CD86, CD54, and MHC II) compared to FC-GFP (p=0.011). Moreover, in a syngeneic mixed lymphocyte reaction, FC-CD40L induced a two-fold higher T-cell proliferation than FC-GFP or FC alone. In addition, FC-CD40L had improved migration to lymphoid tissues, preferentially to spleen, in comparison with FC-GFP (2.8% versus 1.6%). The antitumor effect of FC-CD40L was analyzed in vivo. Mice (n=10 per group) were injected i.v. with 2.5×105 tumor cells and treated with irradiated FC, FC-GFP or FC-CD40L (1×106 cells each) on days 2, 6 and 10 after tumor challenge. 40% of mice treated with FC-CD40L had long-term survival (>120 days). In contrast, all of mice treated with FC or FC-GFP died between days 25 and 35 (p=0.012). In parallel, treatment with mixed cells (not fused DC+ tumor cells), mix transduced with AdvGFP, or mix transduced with AdvCD40L did not provide any significant antitumor effect. We conclude that FCs transduced with AdvCD40L better stimulate in vitro and in vivo immune responses than FC alone and may provide a new strategy for treating patients with multiple myeloma or lymphoma.

Introduction Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism in the clinical activity of rituximab for the treatment of B-cell malignancies. The NK cells, through the activating receptor FcγRIIIa, play a major role in the rituximab-mediated ADCC. We have studied the effect of NK stimulators such IL-15 and CpG oligodeoxynucleotides (ODN) in the enhancement of rituximab-mediated ADCC against B-cell lymphoma. Methods ADCC was performed by standard 51Cr release assays using peripheral blood mononuclear cells (PBMC) purified from 4 normal volunteers by Ficoll-Hypaque gradient centrifugation. Cells were cultured for 18 hours in the presence of human recombinant interleukin 15 (rhIL-15; 10 ng/ml) or CpG ODN (2,5 μg/ml). The B-lymphoma cell line Raji was used as target in the presence of rituximab (10 μg/ml) or human IgG1 as control (10 μg/ml) for 30 min at room temperature. Target cells and effector cells were incubated at different ratios ranging from 1:1 to 30:1, for 4 hours at 37°C. The polymorphism at amino acid 158 of the activating FcγRIIIa receptor (FcγRIIIa V→F) was analyzed in effector cells from normal donors with a polymerase chain reaction (PCR)-based allele-specific restriction analysis assay. Results Lysis of B lymphoma cells treated with rituximab alone was 40% ± 5% (mean ± SD) at the highest ratio, as compared to 12% ± 7% (p Conclusion PBMC cells activated in the presence of IL-15 or CpG ODN become strong effectors in rituximab-mediated ADCC against B cell lymphoma. These results may be useful to improve the therapeutic activity of rituximab.