Despite the production of neutralizing antibodies to hepatitis C virus (HCV), many patients fail to clear the virus and instead develop chronic infection and long-term complications. To understand ...how HCV infection perturbs the antibody repertoire and to identify molecular features of antibody genes associated with either viral clearance or chronic infection, we sequenced the V(D)J region of na#239;ve and memory B cells of 6 persons who spontaneously resolved an HCV infection (SR), 9 patients with a newly diagnosed chronically evolving infection (CE), and 7 healthy donors. In both na#239;ve and memory B cells, the frequency of use of particular antibody gene subfamilies and segments varied among the three clinical groups, especially between SR and CE. Compared to CE, SR antibody genes used fewer VH, D and JH gene segments in na#239;ve B cells and fewer VH segments in memory B cells. SR and CE groups significantly differed in the frequency of use of 7 gene segments in na#239;ve B cell clones and 3 gene segments in memory clones. The nucleotide mutation rates were similar among groups, but the pattern of replacement and silent mutations in memory B cell clones indicated greater antigen selection in SR than CE. Greater clonal evolution of SR than CE memory B cells was revealed by analysis of phylogenetic trees and CDR3 lengths. Pauciclonality of the peripheral memory B cell population is a distinguishing feature of persons who spontaneously resolved an HCV infection. This finding, previously considered characteristic only of patients with HCV-associated lymphoproliferative disorders, suggests that the B cell clones potentially involved in clearance of the virus may also be those susceptible to abnormal expansion.
Anti-carbamylated protein antibodies (anti-CarP Ab) represent a novel kind of autoantibodies specificity detectable in the sera of patients with rheumatoid arthritis (RA). They have been recently ...reported to be associated with increased mortality in Spanish patients with RA. The aim of our study was to compare the incidence mortality rates (IMRs) detected in RA patients from a tertiary Italian center with those reported in other European tertiary centers and to evaluate the putative role of anti-CarP Ab in modulating the low IMR detected in our patients.
Clinical charts of patients consecutively admitted to our center, from January 1, 2008, to December 31, 2014, were retrospectively reviewed. The mortality rate (expressed as the number of deaths in the cohort divided by the number of years of IMR follow-up) and causes of death were assessed at December 31, 2015. Sera of 61 patients, representative of the whole cohort, collected at the time of admission to our center were investigated for the presence and the level of anti-CarP Ab. Demographic and clinical features, mortality rates and prevalence of anti-CarP Ab in our series were compared with those reported in other European cohorts.
We observed 608 patients for a median of 3.51 years. All-cause and cause-specific IMRs in our cohort were significantly lower than the Better Anti-rheumatic Farmaco-therapy and the Spanish cohort, while only all-cause and cardiovascular IMRs were significantly lower in our series with respect to the Leiden Early Arthritis Clinic cohort. Anti-CarP Ab prevalence was significantly lower in our series than in any other European cohorts.
We confirm that the mortality rate is lower in our Italian RA cohort with respect to other European cohorts. Whether the low prevalence of anti-CarP Ab might be responsible for this result awaits to be furtherly investigated.
Despite the production of neutralizing antibodies to hepatitis C virus (HCV), many patients fail to clear the virus and instead develop chronic infection and long-term complications. To understand ...how HCV infection perturbs the antibody repertoire and to identify molecular features of antibody genes associated with either viral clearance or chronic infection, we sequenced the V(D)J region of naïve and memory B cells of 6 persons who spontaneously resolved an HCV infection (SR), 9 patients with a newly diagnosed chronically evolving infection (CE), and 7 healthy donors. In both naïve and memory B cells, the frequency of use of particular antibody gene subfamilies and segments varied among the three clinical groups, especially between SR and CE. Compared to CE, SR antibody genes used fewer VH, D and JH gene segments in naïve B cells and fewer VH segments in memory B cells. SR and CE groups significantly differed in the frequency of use of 7 gene segments in naïve B cell clones and 3 gene segments in memory clones. The nucleotide mutation rates were similar among groups, but the pattern of replacement and silent mutations in memory B cell clones indicated greater antigen selection in SR than CE. Greater clonal evolution of SR than CE memory B cells was revealed by analysis of phylogenetic trees and CDR3 lengths. Pauciclonality of the peripheral memory B cell population is a distinguishing feature of persons who spontaneously resolved an HCV infection. This finding, previously considered characteristic only of patients with HCV-associated lymphoproliferative disorders, suggests that the B cell clones potentially involved in clearance of the virus may also be those susceptible to abnormal expansion.
Purpose: In multiple myeloma (MM), serum beta-2-microglobulin (β2m)-free heavy chains (FHC) of HLA class I has been shown to reflect disease activity. We investigated the possibility of stratifying ...patients with active disease according to FHC and other clinical parameters.
Experimental design: We studied 146 patients with MM, including 100 at diagnosis, 31 in relapse and 15 unresponsive to therapy. Univariate and multivariate analyses were used to assess the prognostic significance of FHC together with continuous variables (age, albumin, creatinine, hemoglobin, erythrocyte sedimentation rate, β2m, calcium, IgM, platelet count) and categorical variables (Durie–Salmon disease stage, gender, bone lesion burden, heavy and light chain isotypes of M-component, clinical status). Survival tree analysis on significant variables was used to develop an MM staging system.
Results: FHC, IgM, platelet count and hemoglobin were independent predictors of prognosis. Survival tree analysis of these variables defined 2 three-risk-group staging systems involving FHC and either IgM or platelet count. Median survival for FHC/IgM stages II and III was 41.5 and 27.8 months, whereas it was not reached for stage I patients (
p
<
0.0001). In the FHC/platelets system, median survival was 93.2 (stage I), 44.1 (stage II) or 27.8 (stage III) months (
p
<
0.0001). Similar results were obtained for the 117 MM patients without renal insufficiency (FHC/IgM
p
<
0.0001; FHC/platelets
p
=
0.001). For the 100 patients at diagnosis, FHC/IgM (
p
=
0.001) was more effective than FHC/platelets (
p
=
0.04).
Conclusions: The independent prognostic markers FHC, IgM and platelets provide two staging systems unaffected by renal insufficiency. Both are effective in evaluating MM patients with active disease.
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 (Ap1-17), which represents, along with Ap17-30, an immunodominant epitope of the protein. Peptide Ap1-17 was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap1-17 antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap17-30. Panning of a phage display peptide library with anti-Ap1-17 antibodies from 2 patients identified two novel, partially overlapping motifs, <5Rx(st)xKP10> and as the result of the alignment of specific phage clone insert sequences. Anti-Ap1-17 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 Ap1-17 antigenic motifs. These data show that anti-CENP-A1-17 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.
Two out of 625 hybridomas constructed with splenocytes from a Balb/c mouse immunized with the murine anti-HLA-DR, DP monoclonal antibody CR11-462 secrete antiidiotypic antibodies. Binding assays with ...a panel of 12 anti-HLA class I, 14 anti-HLA class II, and 9 anti-human melanoma-associated antigen MoAbs showed that the idiotopes recognized by MoAb F3-C25 and F3-B6 are not expressed by any of the tested antibodies, even those which inhibit the binding of MoAb CR11-462 to cultured B lymphoid cells. Crossblocking experiments showed that the two idiotopes are distinct and spatially distant. Both idiotopes require the association of heavy and light chain of MoAb CR11-462 for their expression. The idiotope recognized by MoAb F3-B6 is an alpha idiotope, since it is located outside the antigen combining site of MoAb CR11-462. The idiotope recognized by MoAb F3-C25 is a gamma idiotope, since it is antigen inhibitable but does not possess an internal image of the antigen. Interestingly the MoAb F3-C25 displays a differential inhibitory effect on the binding of MoAb CR11-462 to lymphoid cells that express different HLA phenotypes or share the HLA-DRw6 allospecificity. These results suggest that antiidiotypic antibodies may sharpen our ability to dissect the heterogeneity of HLA class II antigens.
We previously showed that serum beta‐2‐microglobulin (β2m)‐free human leucocyte antigen (HLA) class I heavy chain (FHC) levels were increased in MM and correlate with disease activity. The present ...investigation, carried out in 124 multiple myeloma patients, studied the expression of the three size variants of FHC, namely the 42 kDa intact heavy chain (A variant, AV), released through a shedding process, and the truncated FHC (tFHC) 39 kDa (BV) and 36–35 kDa (CV) released by means of membrane‐type metalloprotease activity. The increase in FHC correlated with a high expression percentage of BV (r = 0·32, P = 0·0002) and tFHC (r = 0·42, P < 0·0001), which could help to discriminate multiple myeloma from monoclonal gammopathy of undetermined significance (tFHC mean ratio = 3·2; Mann–Whitney U‐test, P < 0·0001). tFHC levels highly correlated with other disease activity markers, namely haemoglobin (r = −0·35; Spearman's rank, P = 0·0001), percentage of bone marrow plasma cells (r = 0·4, P < 0·0001) and β2m levels (r = 0·36, P < 0·0001), while only the last barely correlated (r = 0·2, P = 0·03) with AV. Finally, the 0·4, 0·57 and 0·71 mg/l BV, tFHC and (to a lesser extent) FHC cut‐off values divided patients into two groups with different survival curves (P = 0·0005, P = 0·0025 and P = 0·04 respectively). These data are in favour of a correlation between disease aggressiveness and cleavage of these variants by membrane‐type metalloprotease enzymes.
In multiple myeloma (MM), serum beta-2-microglobulin (beta2m)-free heavy chains (FHC) of HLA class I has been shown to reflect disease activity. We investigated the possibility of stratifying ...patients with active disease according to FHC and other clinical parameters.
We studied 146 patients with MM, including 100 at diagnosis, 31 in relapse and 15 unresponsive to therapy. Univariate and multivariate analyses were used to assess the prognostic significance of FHC together with continuous variables (age, albumin, creatinine, hemoglobin, erythrocyte sedimentation rate, beta2m, calcium, IgM, platelet count) and categorical variables (Durie-Salmon disease stage, gender, bone lesion burden, heavy and light chain isotypes of M-component, clinical status). Survival tree analysis on significant variables was used to develop an MM staging system.
FHC, IgM, platelet count and hemoglobin were independent predictors of prognosis. Survival tree analysis of these variables defined 2 three-risk-group staging systems involving FHC and either IgM or platelet count. Median survival for FHC/IgM stages II and III was 41.5 and 27.8 months, whereas it was not reached for stage I patients (p<0.0001). In the FHC/platelets system, median survival was 93.2 (stage I), 44.1 (stage II) or 27.8 (stage III) months (p<0.0001). Similar results were obtained for the 117 MM patients without renal insufficiency (FHC/IgM p<0.0001; FHC/platelets p=0.001). For the 100 patients at diagnosis, FHC/IgM (p=0.001) was more effective than FHC/platelets (p=0.04).
The independent prognostic markers FHC, IgM and platelets provide two staging systems unaffected by renal insufficiency. Both are effective in evaluating MM patients with active disease.
The mouse anti-idiotypic mAb (mAb2) 16D7 recognizes the paratope (combining site-associated epitope) of the syngeneic anti-human CD4 mAb HP2/6 (mAb1), a down-modulator of T cell function. 16D7 mimics ...CD4 in xenogeneic settings in humans and can thus be used to target CD4
+ T cells for therapeutic purposes. To define the minimum structural requirement for CD4 mimicry, 16D7 isolated L and H chains were tested for their ability to inhibit mAb1 binding to either CD4 Ag or to mAb2. 16D7 L only specifically inhibited these interactions. Alignment of 16D7 L variable region sequence with that of CD4 defined peptides L2, L3 and L7 with at least 68% of similarity and 30% of identity to exposed regions of CD4. Furthermore, peptides L1, L4, L5, L6 and L8 were selected from a set of 10-mer overlapping peptides (covering the FW and CDR regions of 16D7 L) because of their reactivity with mAb1. Only the synthesized L2 reacted with HP2/6 in slot dot assay and inhibited mAb2–mAb1 interaction. Immunization of BALB/c mice with 16D7, 16D7-F(ab’)
2, 16D7 L and L2, resulted in the production of CD4-specific Ab3 only in mice immunized with 16D7 and its F(ab’)
2 fragments. The lack of CD4 reactivity with sera elicited with 16D7 L and L2 was not due to their lack of immunogenicity since both sera reacted with the corresponding immunogen and the former also inhibited the binding of mAb1 to 16D7. The results suggest that CD4 mimicry by 16D7 is based on a conformational epitope and L2 is only one of the HP2/6-specific contact points of 16D7.
mAb HC-10 loses its reactivity with HLA class I (HLA-I) H chain (HC) following its association with beta(2)-microglobulin (beta(2)m). Furthermore, the HC-10 defined epitope appears to be involved in ...the pathogenesis of spondyloarthropathies, because HC-10 reduced their incidence in HLA-B27(+)beta(2)m degrees /MHC class II knockout mice. This study has characterized the determinant recognized by HC-10. Panning of a phage display peptide library with HC-10 resulted in isolation of the motif PxxWDR, which could be aligned with P57, W60, D61, and R62 of the first domain of the HLA-I HC allospecificities reactive with HC-10. The (55)EGPEYWDR(N/E)T(64) (p-1) is the shortest motif-bearing peptide that reacts with HC-10 and inhibits its binding to soluble HLA-B7 HC, irrespective of whether N (p-1a) or E (p-1b) is present at position 63. By contrast, HC-10 did not react with six additional peptides, each bearing motif amino acid substitutions present in HC-10-not-reactive HLA-I allospecificities. The p-1-derived Qp-1, synthesized with the additional conserved Q54, which displays the highest in vitro reactivity with HC-10, was the only one to induce in mice IgG resembling HC-10 in their fine specificity. Mapping of the HC-10-defined determinant suggests that the lack of mAb reactivity with beta(2)m-associated HLA-I HC is caused by blocking by the peptide in the groove of beta(2)m-associated HLA-I HC, though a role of HC conformational changes following its association with beta(2)m cannot be excluded. This information contributes to our understanding of the molecular basis of the antigenic profiles of beta(2)m-free and beta(2)m-associated HLA-I HC and may serve to develop active specific immunotherapy of spondyloarthropathies.
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