Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and ...identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.
Background. Absence of acquired protective immunity in endemic areas children leads to higher susceptibility to severe malaria. To investigate the involvement of regulatory process related to ...self-reactivity, we evaluated potent changes in auto-antibody reactivity profiles in children and older subjects living in malaria-endemic zones comparatively to none-exposed healthy controls. Methods. Analysis of IgG self-reactive footprints was performed using Western blotting against healthy brain antigens. Plasmas of 102 malaria exposed individuals (MEIs) from endemic zone, with or without cerebral malaria (CM) were compared to plasmas from non-endemic controls (NECs). Using linear discriminant and principal component analysis, immune footprints were compared by counting the number, the presence or absence of reactive bands. We identified the most discriminant bands with respect to age and clinical status. Results. A higher number of bands were recognized by IgG auto-antibodies in MEI than in NEC. Characteristic changes in systemic self-IgG-reactive repertoire were found with antigenic bands that discriminate Plasmodium falciparum infections with or without CM according to age. 8 antigenic bands distributed in MEI compared with NEC were identified while 6 other antigenic bands were distributed within MEI according to the age and clinical status. Such distortion might be due to evolutionary processes leading to pathogenic/protective events.
Abstract In the present study we showed that transitional B cells of patients with clinically isolated syndrome (CIS) and relapsing–remitting multiple sclerosis (RR–MS) are reduced in the peripheral ...blood (PB) (5.5- and 3.7-fold, respectively). In addition, these cells appeared to up-regulate different integrins (α4 and β1). These observations were associated with a primed cellular status, confirmed by an increased proportion of circulating CD80+ transitional B cells. Interestingly, these results correlate with presence of transitional B cells in the CSF. Furthermore, these cells were absent in the CSF of individuals with other inflammatory neurological disease, and their levels in paired PB and CD80 expression were normal. Altogether, our data revealed that a differential primed status of transitional B cells is a characteristic feature of early phases of MS disease, and this functional status is associated with the ability of these cells to cross the blood–CSF barrier.
We sequentially analyzed the serum IgG response against normal mouse brain during experimental autoimmune encephalomyelitis in SJL/J mice injected with CFA,
Bordetella pertussis toxin (BPT) and ...proteolipid protein 139–151 peptide, compared with mice that received CFA and BPT or were uninjected. Dynamic changes were observed from day 0 to day 28 in the 3 groups. Six highly discriminant antigenic bands (
κ
=
0.974) were identified. Three non-myelin proteins were characterized (mitochondrial aconitase hydratase 2, phosphoglycerate mutase 1, brain specific pyruvate deshydrogenase). The IgG response against two of them was less frequent in EAE whereas it was associated with multiple sclerosis in our previous work.
Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and ...identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.
Abstract We reduced EAE severity by using two anti-allergic drugs. A control group of mice received i.p. injections of PBS as vehicle while a further two groups were treated either with pyrilamine, a ...histamine receptor 1 antagonist or with CV6209, a platelet activating factor receptor antagonist. Our results showed that the blockade of the responses to both histamine and PAF leads together to a decline in clinical signs of EAE and significant changes in the serum IgG recognition of some healthy brain antigenic targets. We characterized two discriminant antigens: internexin neuronal intermediate filament protein, and malate dehydrogenase 1, which were able to clearly distinguish untreated mice from treated mice. Their role as potent targets in pathogenic and/or neuroprotective processes is discussed.
Objective
To characterize discriminant human brain antigenic targets in patients with neuropsychiatric systemic lupus erythematosus (NPSLE), using a standardized immunoproteomic approach.
Methods
...Self‐IgG reactivity against normal and injured human brain tissues was studied by Western blotting of sera from 169 subjects, 16 patients with NPSLE, 12 patients with SLE without neuropsychiatric manifestations (non‐NPSLE), 32 patients with Sjögren's syndrome with or without central nervous involvement, 82 patients with multiple sclerosis, and 27 healthy subjects. A proteomic approach was then applied to characterize discriminant antigens identified after comparisons of all patterns.
Results
The serum self‐IgG reactivity patterns against human brain tissue differed significantly between patients with NPSLE and the control groups. Four normal brain antigenic bands were specifically or preferentially recognized by sera from NPSLE patients (p240, p90, p77, and p24). Protein band p240 was characterized as microtubule‐associated protein 2B (MAP‐2B), p77 as Hsp70–71, and p24 as triosephosphate isomerase. Protein band p90 was not characterized. In contrast, 1 other protein band (p56, characterized as septin 7) was never recognized by sera from NPSLE patients but was recognized by a majority of sera from non‐NPSLE patients.
Conclusion
Our findings show that the immunoproteomic approach is a reliable method for assessing serum self‐IgG reactivities against human brain tissue in NPSLE. Our characterization of some of the identified discriminant antigens, such as MAP‐2B, triosephosphate isomerase, and septin 7, suggests that the stability of neuronal microtubules might be involved in the pathophysiology of NPSLE.
Radiotherapy is the standard salvage treatment after radical prostatectomy. To date, the role of androgen deprivation therapy has not been formally shown. In this follow-up study, we aimed to update ...the results of the GETUG-AFU 16 trial, which assessed the efficacy of radiotherapy plus androgen suppression versus radiotherapy alone.
GETUG-AFU 16 was an open-label, multicentre, phase 3, randomised, controlled trial that enrolled men (aged ≥18 years) with Eastern Cooperative Oncology Group performance status of 0 or 1, with histologically confirmed adenocarcinoma of the prostate (but no previous androgen suppression or pelvic radiotherapy), stage pT2, T3, or T4a (bladder neck involvement only) and pN0 or pNx according to the tumour, node, metastasis (TNM) staging system, whose prostate-specific antigen (PSA) concentration increased from 0·1 ng/mL to between 0·2 ng/mL and 2·0 ng/mL after radical prostatectomy, without evidence of clinical disease. Patients were assigned through central randomisation (1:1) to short-term androgen suppression (subcutaneous injection of 10·8 mg goserelin on the first day of irradiation and 3 months later) plus radiotherapy (3D conformal radiotherapy or intensity modulated radiotherapy of 66 Gy in 33 fractions, 5 days a week for 7 weeks) or radiotherapy alone. Randomisation was stratified using a permuted block method (block sizes of two and four) according to investigational site, radiotherapy modality, and prognosis. The primary endpoint was progression-free survival in the intention-to-treat population. This post-hoc one-shot data collection done 4 years after last data cutoff included patients who were alive at the time of the primary analysis and updated long-term patient status by including dates for first local progression, metastatic disease diagnosis, or death (if any of these had occurred) or the date of the last tumour evaluation or last PSA measurement. Survival at 120 months was reported. Late serious adverse effects were assessed. This trial is registered on ClinicalTrials.gov, NCT00423475.
Between Oct 19, 2006, and March 30, 2010, 743 patients were randomly assigned, 374 to radiotherapy alone and 369 to radiotherapy plus goserelin. At the time of data cutoff (March 12, 2019), the median follow-up was 112 months (IQR 102–123). The 120-month progression-free survival was 64% (95% CI 58–69) for patients treated with radiotherapy plus goserelin and 49% (43–54) for patients treated with radiotherapy alone (hazard ratio 0·54, 0·43–0·68; stratified log-rank test p<0·0001). Two cases of secondary cancer occurred since the primary analysis, but were not considered to be treatment related. No treatment-related deaths occurred.
The 120-month progression-free survival confirmed the results from the primary analysis. Salvage radiotherapy combined with short-term androgen suppression significantly reduced risk of biochemical or clinical progression and death compared with salvage radiotherapy alone. The results of the GETUG-AFU 16 trial confirm the efficacy of androgen suppression plus radiotherapy as salvage treatment in patients with increasing PSA concentration after radical prostatectomy for prostate cancer.
The French Health ministry, AstraZeneca, la Ligue Contre le Cancer, and La Ligue de Haute-Savoie.
We evaluated the presence of human herpesvirus 8 (HHV‐8) infection among groups of Amerindians in French Guiana. The overall prevalence of antibodies against lytic HHV‐8 antigens was 23.0% (180/781), ...increasing from 18.4% in children <6 years old to ∼30% in older persons (>45 years). Seroprevalence was higher in Amerindians living in remote localities than it was in those living in the coastal region. Analysis of a 725–base pair fragment of the K1 gene amplified from DNA from a Wayampi Amerindian showed that the virus belonged to molecular subtype E, which has hitherto been found in only a few Amerindians in Brazil and Ecuador.
To strengthen active dengue surveillance in Saint Martin and Saint Barthélemy, two French Caribbean islands, we evaluated the epidemiological usefulness of collecting blood samples from NS1-positive ...dengue patients on filter paper to identify the dengue serotypes circulating in these regions during a 27-month period. This approach allowed dengue serotypes to be identified by reverse transcriptase-polymerase chain reaction in 90.1% of the total set of 666 samples analyzed and, in 95.5% of the samples collected during the acute phase of the disease. This prospective virological surveillance using blood samples absorbed onto filter paper, which were stored at 4°C and shipped at ambient temperature to a specialized laboratory for analysis, allowed us to avoid the logistic and financial costs associated with shipping frozen venous blood samples. This surveillance system offers a low-cost alternative for reinforcing dengue prevention in areas where specialized laboratories do not exist, notably by facilitating the early detection of potentially new dengue serotypes.
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