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IntroductionBoth erosions and osteoporosis are present in rheumatoid arthritis and are related to RANK-L pathway activation. The aim of the study was to evaluate the relationship between erosion and bone mineral density (BMD) in RA and whether it can be driven by autoimmunity.Patients and methodsPatients followed in the Department of Rheumatology between January 2008 and May 2019 satisfied the 1987 ACR or 2010 ACR-EULAR criteria. Erosions were evaluated by the modified Sharp/van der Heidje erosion score (SHSe) on radiographs and bone mineral density (BMD) in g/cm2 and by the T-score at the hip on DXA. The presence and titers of ACPA as well as rheumatoid factor (RF) and anti-nuclear antibodies (ANAs) were recorded at intervals of less than 2 years for both DXA and radiography.ResultsA total of 149 patients met the inclusion criteria. A total of 61.1% were ACPA positive, 79.9% were erosive and 10.7% had a hip T-score ≤-2.5. ACPA status but not titers was associated with a higher erosion score (63.0 (53.2) for ACPA + vs. 45.5 (44.1) for ACPA – (p= 0.04)). ACPA titers were associated with lower BMD at the hip (value -0.216; p=0.01) but not with T-score. A higher erosion score was associated with a lower BMD (R2: 0,049 and value: -0.222; p=0.009) and T-score (R2: 0,158 and value -0.397; p

Objectives Clinical significance of anti-Ro52 antibodies in connective tissue diseases (CTD) is controversial. Anti-Ro52 antibodies might be associated with a more severe CTD phenotype, especially interstitial lung disease (ILD). The aims of this study were to evaluate ILD prevalence and severity, the prevalence of micro- or macroangiopathy and CTD-associated cancers in CTD with anti-Ro52 antibodies. Methods CTD patients with anti-Ro52 antibody screening by immunoblot at diagnosis were enrolled. Two groups were retrospectively formed according to the presence of anti-Ro52 antibodies with an unbiased 1:1 matching on CTD types. Unsupervised multiple correspondence analysis and hierarchical clustering analysis were used to aggregate anti-Ro52 positive patients in subgroups. Results 408 CTD patients were included. Anti-Ro52 antibodies were detected in 33 % of CTD patients. Anti-Ro52 antibodies were associated with ILD at CTD diagnosis (47.8% vs. 23.0%, OR 3.3 95% IC 1.4 to 8.0, p = 0.008), even after adjusting for the presence of anti-Ro60 antibodies, especially in patients with antisynthetase syndrome, primary Sjogren syndrome and systemic sclerosis. Micro- or macroangiopathy was more frequent in anti-Ro52 positive CTD patients (18.6% vs. 9.7%, p = 0.02) and CTD patients with anti-Ro52 antibodies experienced more frequent relapses and required more immunosuppressive drugs. Clusters 4 and 5 identified anti-Ro52 positive CTD patients with severe ILD and with clinical features of systemic sclerosis or antisynthetase syndrome respectively. Conclusions We found that anti-Ro52 antibodies were independently associated with ILD in CTD patients irrespective of CTD type. Anti-Ro52 antibodies could be associated with severity and a more relapsing disease course in CTD patients.

Resume Les lymphocytes T a recepteur antigenique chimerique (CAR-T cells) representent une innovation majeure offrant aux patients une nouvelle possibilite therapeutique pour des maladies graves. L’injection des CAR-T cells s’accompagne d’un passage de ces cellules dans la circulation sanguine. Cet article rapporte une observation de detection morphologique et de suivi cytometrique de ces cellules.

The new coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has recently put the world under stress, resulting in a global pandemic. Currently, there are no approved treatments or vaccines, and this severe respiratory illness has cost many lives. Despite the established antimicrobial and immune-boosting potency described for honey, to date there is still a lack of evidence about its potential role amid COVID-19 outbreak. Based on the previously explored antiviral effects and phytochemical components of honey, we review here evidence for its role as a potentially effective natural product against COVID-19. Although some bioactive compounds in honey have shown potential antiviral effects (i.e., methylglyoxal, chrysin, caffeic acid, galangin and hesperidinin) or enhancing antiviral immune responses (i.e., levan and ascorbic acid), the mechanisms of action for these compounds are still ambiguous. To the best of our knowledge, this is the first work exclusively summarizing all these bioactive compounds with their probable mechanisms of action as antiviral agents, specifically against SARS-CoV-2.

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Background and Aims Histological healing may be the ultimate therapeutic goal in ulcerative colitis [UC]. We investigated, for the first time, the association between vedolizumab trough levels and histological healing in UC. Methods This is a single-centre retrospective cohort study including all consecutive UC patients on vedolizumab maintenance therapy who had a histological evaluation blindly to clinical data and underwent therapeutic drug monitoring, between June 2014 and March 2018. Per-event analysis was performed. Histological healing was defined as a Nancy histological index ≤1. Results Thirty-five histological samples were analysed. Median [interquartile range] vedolizumab trough levels were higher in the group with histological healing (31.5 [25–49.1] μg/mL) compared with the group without histological healing (15 [9–26.6] μg/mL, p = 0.02). The higher vedolizumab trough level quartiles tended to be associated with greater rates of histological healing [p = 0.10]. A cut-off vedolizumab trough level of 25 μg/mL predicted histological healing with an accuracy of 74% and an area under the receiver operating curve of 0.62 [95% confidence interval 0.58–0.92, p = 0.004]. Bivariate analysis identified a vedolizumab trough level ≥25 µg/mL [p = 0.006], a partial Mayo score ≤1 [p = 0.008], C-reactive protein level Conclusions Histological healing was associated with higher vedolizumab trough levels during maintenance therapy in UC. A vedolizumab trough level threshold of 25 μg/mL proved most optimal to predict histological healing according to the Nancy histological index. Confirmation of these data in larger, independent cohorts is needed.

The objective of this study is to assess the prevalence, localization, and severity of bone erosions on radiography (RX) and ultrasonography (US) according to ACPA status in patients with rheumatoid arthritis (RA). 78 patients with ACPA-positive (ACPA+) RA and 30 patients with ACPA-negative (ACPA−) RA fulfilling the ACR 1987 and/or ACR/EULAR 2010 criteria were consecutively included. On RX, a modified Sharp erosion score (SHSe) was evaluated by two blinded readers and one adjudicator for discordant cases (number of eroded joints ≤ three). On US, erosions were scored on six bilateral joints (MCP2, 3, 5; MTP2, 3, 5) with a four-point scale to calculate the total US score for erosions (USSe). The mean total SHSe and USSe were 3.7 and 4.4 times higher in the ACPA+ group than in the ACPA− group, respectively (P

CAR-T cells are a new possibility for care poor prognostic conditions. CAR-T cells injection lead to T chimeric cells circulation into the blood. Here is reported the cytomorphologic and immunophenotypic features of CAR-T cells in the peripheral blood.

One third of pediatric IBD patients who initially respond to infliximab (IFX) lose that response over time. This retrospective study, including 62 children treated with IFX from 2004 to 2017, aimed to identify factors associated with clinical flare. Ulcerative colitis, extreme body mass index, and lowest IFX trough levels were associated with clinical flare in the whole population. In Crohn disease patients, perianal disease was pejorative, while location proximal to ligament of Treitz was protective. Underweight patients probably correspond to the most severe cases who are more likely to relapse, with hypoalbuminemia responsible for lower systemic IFX availability. Obesity probably induces higher IFX clearance.

Background Despite the critical roles of Th1-polarised CD4+ T cells in cancer immunosurveillance, the translation of their potential to clinical use remains challenging. Here, we investigate the clinical relevance of circulating antitumor Th1 immunity in non-small cell lung cancer (NSCLC). Methods The circulating antitumor Th1 response was assessed by the ELISpot assay in 170 NSCLC patients using a mixture of HLA class II-restricted peptides from telomerase (TERT). Phenotyping of blood immune cells was performed by flow cytometry. Results TERT-reactive CD4 T-cell response was detected in 35% of NSCLC patients before any treatment. Functional analysis showed that these cells were effector memory and Th1 polarised capable to produce effector cytokines, such as IFN-γ, TNF-α and IL-2. The presence of anti-TERT Th1 response was inversely correlated with the level of exhausted PD-1+/TIM-3+CD4 T cells. The level of these two immune parameters differentially affected the survival, so that increased level of anti-TERT Th1 response and low rate of exhausted PD-1+TIM-3+CD4+ T cells were associated with a better prognosis. Conclusions Systemic anti-TERT Th1 response plays a strong antitumor protective role in NSCLC. This study underlines the potential interest of monitoring circulating antitumor Th1 response for patients’ stratification and therapy decision.

International audience; BACKGROUND:Despite aggressive regimens, the clinical outcome of head and neck squamous cell carcinoma remains poor. The detection of circulating tumor cells could potentially improve the management of patients with disseminated cancer, including diagnosis, treatment strategies, and surveillance. Currently, CellSearch(®) is the most widely used and the only Food and Drug Administration-cleared system for circulating tumor cells detection in patients with metastatic breast, colorectal, or prostate cancer. In most cases of head and neck squamous cell carcinoma, only low counts of circulating tumor cells have been reported.CASE PRESENTATION:A 56-year-old white male with no particular medical history, was diagnosed with a squamous cell carcinoma of oral cavity. According to the imaging results (computed tomography and (18)F-fluorodeoxyglucose positron emission tomography / computed tomography) and panendoscopy, the TNM staging was classified as T4N2M0. A non-interruptive pelvimandibulectomy was conducted according to the multidisciplinary meeting advices and the postoperative observations were normal. The patient complained of a painful cervical edema and a trismus 6 weeks after the surgery. A relapse was found by computed tomography and the patient died two weeks later. The search for circulating tumor cells in peripheral venous blood by using the CellSearch(®) system revealed a very high count compared with published reports at three time points (pre-operative: 400; intra-operative: 150 and post-operative day 7: 1400 circulating tumor cells). Of note, all detected circulating tumor cells were epidermal growth factor receptor negative.CONCLUSION:We report here for the first time a rare case of oral squamous cell carcinoma with extremely high circulating tumor cells counts using the CellSearch(®) system. The absolute number of circulating tumor cells might predict a particular phase of cancer development as well as a poor survival, potentially contributing to a personalized healthcare.

EA MERS; Rationale: Although the outcome of sepsis benefits from the prompt administration of appropriate antibiotics on correct diagnosis, the assessment of infection in critically ill patients is often a challenge for clinicians. In this setting, simple biomarkers, especially when used in combination, could prove useful. Objectives: To determine the usefulness of combination biomarkers to diagnose sepsis. Methods: Three hundred consecutive patients were enrolled to construct a biologic score that was next validated in an independent prospective cohort of 79 critically ill patients from another center. Measurement and Main Results: Plasma concentrations of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and procalcitonin (PCT) were assayed, and the expression of the high-affinity immunoglobulin-Fc fragment receptor I (Fc gamma RI) CD64 on neutrophils (polymorphonuclear [PMN] CD64 index) in flow cytometry was measured. A "bioscore" combining these biomarkers was constructed. Serum concentrations of PCT and sTREM-1 and the PMN CD64 index were higher in patients with sepsis compared with all others (P < 0.001 for the three markers). These biomarkers were all independent predictors of infection, the best receiver-operating characteristic curve being obtained for the PMN CD64 index. The performance of the bioscore, better than that of each individual biomarker, was externally confirmed in the validation cohort. Conclusions: This prospective study, including inceptive and validation cohorts of unselected intensive care unit patients, demonstrates the high performance of a bioscore combining the PMN CD64 index together with PCT and sTREM-1 serum levels in diagnosing sepsis in the critically ill patient.

Laboratory predictors of severe forms of dengue virus (DENV) infection are needed. These clinical forms seem to be associated with high viremia, stressing the importance of immune responses, which could involve dendritic cells (DC). Yet, very few studies have evaluated DC after DENV infection. We assessed peripheral blood DC subset numbers in mild and severe forms of dengue in 44 patients, older than 15 years old, infected with serotypes DENV-2, 3 or 4. Patients were divided in high, intermediate and no detectable viremia according to results of molecular biology amplification of DENV. Serological status of anti-DENV IgG or IgM determined primary or secondary infections. Plasmacytoid and myeloid DC absolute and relative numbers were reduced in infected patients when compared to an age-matched healthy control group, but no significant differences in DC numbers were observed between mild or severe forms of disease. A severe disease was more frequent in patients infected with DENV-2 serotype and with secondary infection but no significant differences in DC subset numbers were found related to these variables. Viremia levels did not correlate to disease severity yet were associated to lower DC numbers. Plasmacytoid DC numbers were significantly lower in high and intermediate viremia groups compared to non-infected controls, but not in no detectable viremia patients. Myeloid DC numbers were also significantly lower than controls, even in no detectable viremia patients. These results confirm that circulating DC subset numbers are reduced after DENV infection, although this is not a biomarker of severe forms of dengue in adults.

International audience; Adoptive antiviral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is becoming an alternative treatment for viral infection after hematopoietic stem cell transplantation. The T memory stem cell (TSCM) subset was recently described as exhibiting self-renewal and multipotency properties which are required for sustained efficacy in vivo. We wondered if such a crucial subset for immunotherapy was present in VSTs. We identified, by flow cytometry, TSCM in adenovirus (ADV)-specific interferon (IFN)-γ+ T cells before and after IFN-γ-based immunomagnetic selection, and analyzed the distribution of the main T-cell subsets in VSTs: naive T cells (TN), TSCM, T central memory cells (TCM), T effector memory cell (TEM), and effector T cells (TEFF). In this study all of the different T-cell subsets were observed in the blood sample from healthy donor ADV-VSTs, both before and after IFN-γ-based immunomagnetic selection. As the IFN-γ-based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets, we observed that TEM was always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation and especially after expansion in vitro. Comparing T-cell subpopulation profiles before and after in vitro expansion, we observed that in vitro cell culture with interleukin-2 resulted in a significant expansion of TN-like, TCM, TEM, and TEFF subsets in CD4IFN-γ T cells and of TCM and TEM subsets only in CD8IFN-γ T cells. We demonstrated the presence of all T-cell subsets in IFN-γ VSTs including the TSCM subpopulation, although this was weakly selected by the IFN-γ-based immunomagnetic selection system.

Several techniques have been developed to detect circulating tumor cells (CTC) in patients with head and neck squamous cell carcinoma (HNSCC), but their diagnostic and prognostic value are not yet fully established. A computerized retrieval of literatures was conducted without time restrictions using the electronic database in December 2014. Diagnostic accuracy variables were pooled and analyzed by the Meta-DiSc software. Engauge Digitizer and Stata software were used for pooled survival analysis. Twenty-two retrieved studies were eligible for systematic review, of which 9 conformed for the diagnostic test meta-analysis and 5 for the prognostic analysis. Subgroup analysis showed 24.6% pooled sensitivity and 100% pooled specificity of detections by using positive selection strategy, which moreover presented low heterogeneity. The presence of CTC was significantly associated with shorter disease free survival (DFS, HR 4.62, 95% CI 2.51–8.52). In conclusion, current evidence identifies the CTC detection assay as an extremely specific, but low sensitive test in HNSCC. Also, the presence of CTC indicates a worse DFS.

Epigenetic modifications of chromatin may play a role in maintaining viral latency and thus persistence of the human T-lymphotropic virus type 1 (HTLV-1), which is responsible for HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A major determinant of disease progression is increased peripheral blood proviral load (PVL), possibly via the accumulation of infected cells in the central nervous system (CNS) creating a damaging inflammatory response. Current therapeutic approaches that focus on reducing either cell proliferation, viral replication, or tissue invasion are still unsatisfactory. Contrasting with these inhibitory strategies, we evaluated the efficacy of a novel approach aimed, paradoxically, at activating viral gene expression to expose virus-positive cells to the host immune response. We used valproate (VPA), a histone deacetylase inhibitor that has been used for decades as a chronic, safe treatment for epileptic disorders. Based on in vitro and in vivo data, we provide evidence that transient activation of the latent viral reservoir causes its collapse, a process that may alleviate the condition of HAM/TSP. This represents the first such approach to treating HAM/TSP, using gene activation therapy to tilt the host-pathogen balance in favor of an existing antiviral response. This trial is registered at http://clinicaltrials.gov/as no. NCT00519181.

Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4(+)CD25(+)) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-gamma-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-gamma-driven responses.

Background Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Methods Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells, and CD4+ absolute counts (ACs). Results Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08, and 0.18 for CD3%, CD4%, CD8%, and CD4 ACs, respectively. Discussion In-house follow-up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 International Clinical Cytometry Society

Objectives The diagnosis of solid cancer leptomeningeal metastasis (LM) relies on the cytology of cerebrospinal fluid (CSF) and/or imaging evidence of neuraxis, yet both lack sufficient sensitivity. The utility of the CellSearch ® , an FDA -approved technology, in assessing CSF tumor cell (CSFTC) was evaluated here in the diagnosis and treatment of patients with lung cancer-related LM. Materials and methods In 18 patients with magnetic resonance imaging (MRI) confirmed LM due to lung cancer, 5mL of CSF were collected in CellSave ® preservative tubes, which allow performing the assay within 96h after sampling. Using a previously adapted CellSearch ® method, we detected, visualized and enumerated CSFTCs and compared the results with conventional cytology. In 3 patients, tumor cells were evaluated sequentially to explore the predictive role of CSFTCs enumeration in the treatment response monitoring. Results CSFTCs were disclosed in 14 of 18 MRI confirmed LM samples (median 785CSFTCs/5mL CSF, range 1 to >20,000), yielding a sensitivity of 77.8%, compared with 44.4% for conventional cytology. CSFTC clusters were observed in 12 patients, similar to those previously described in blood as circulating tumor microemboli (CTM), and enumerated sequentially with reproducible results, which did not necessarily correlate with response to treatment. Conclusion The CellSearch ® technology, applied to limited sample volumes and allowing delayed processing, could be of great interest in the diagnosis of LM in lung cancer patients.

Cell-based therapies are promising approaches to treat uncontrolled pathologies, such as tumours. Apoptotic tumour cells have recently been proposed as a source of tumour-associated antigens to stimulate an efficient immune response. However, a complex relationship exists between apoptosis and the immune system. In this review, the different factors that may influence immune responses against apoptotic cells are detailed and discussed in the light of recent publications. These factors include the nature of the phagocytes and the receptors involved in apoptotic cell uptake, as well as the environment in which cells are dying. A possible distinction between apoptosis and necrosis by immune system sentinels adds a further level of complexity. The potential use of the immunomodulatory properties associated with apoptosis to favour engraftment and induce tolerance in transplantation is then discussed. In conclusion, this review will suggest appropriate conditions to efficiently and safely use apoptotic cells as a new cell therapy product.

Background Gene-transfer techniques are commonly employed for both in vitro and in vivo studies. However, modifications of the target cell following the introduction of the gene of interest are not often examined. These modifications can alter the immunogenicity and/or the susceptibility of the target cell to apoptosis and may produce unwanted consequences in vivo. Methods Gene transfer into the murine fibroblastic Ψ-CRIP packaging cellline was performed using calcium phosphate precipitation, cationic liposome–DNA complexes or a retroviral RNA-mediated method. After gene transfer, Fas expression, cytokine production, and sensitivity to Fas ligand (FasL)-mediated death were assessed. Results Following transfection of a FasL expression vector by calcium phosphate precipitation, an unexpected increase was observed in apoptotic cell death in previously Fas-resistant Ψ-CRIP cells. This apoptosis was due to Fas upregulation and an increase of sensitivity to FasL-mediated death. Other plasmids coding non-cytotoxic factors also modulated this apoptotic pathway. Theco-stimulatory molecule CD80 was also upregulated. Exposure to naked DNA alone elicited the same response. The effect was not dependent on the methylation status of exogenous DNA, but was found to be dependent on the target cell type and might be avoided by the use of an RNA-mediated retroviral system. Conclusions Plasmid transfection or simple exposure to naked DNA can increase sensitivity to apoptosis. The generation of FasL packaging cell lines is therefore limited by an increase in FasL/Fas-mediated apoptosis. These findings should be considered when using genetically modified transplantable cells in order to prevent elimination by host cytotoxic cells and in particular when cells are engineered using FasL. Copyright © 2001 John Wiley & Sons, Ltd.

Introduction Background. The clinical and immunological relevance of a positive B-cell flow-cytometry (B-FCXM ) Pretransplant crossmatch ( XM ) detection of anticrossmatch in renal transplantation is still donor IgG antibodies to HLA class I molecules contracontroversial. indicates renal transplantation. The presence of such Methods. We retrospectively analysed 145 consecutive antibodies is associated with hyperacute graft rejection cadaveric renal transplantations performed from as well as reduced graft survival. Presently, comMay 1991 to September 1995 in our institution. All plement-dependent cytotoxicity (CDC ) test remains grafts were transplanted following a negative IgG the standard pretransplant crossmatch technique. T-cell complement-dependent cytotoxicity crossmatch However, graft rejection and overall poor graft survival ( T-CDCXM ). Concomitantly to CDCXM, B-cell and can be observed despite a negative CDCXM. T-cell FCXM were performed and results were Development of alternative methods for the detection expressed as a mean fluorescence index ( FI ). Two of recipient serum antibodies directed against donor groups were compared: 116 recipients grafted with a determinants is therefore necessary. Several teams pernegative B-FCXM vs a group of 19 patients grafted form the anti-globulin assay, increasing the sensitivity with a positive B-FCXM. of the standard CDC by the addition of a complementResults. The two groups were similar for length of fixing anti-human IgG. Flow-cytometry crossmatch cold ischaemia, donor and recipient’s age and degree ( FCXM ) is a more sensitive technique that allows the of HLA mismatching. The proportion of patients with detection of low titres of both complement-dependent pre-transplant anti-HLA class I antibodies or a retrans- and -independent anti-donor antibodies, and is thereplantation was significantly increased in the positive fore increasingly used in addition to the standard CDC B-FCXM group vs the negative B-FCXM group. test [reviewed in 1]. FCXM has been reported to be at Recipient survival at 48 months was not significantly least as sensitive as the anti-globulin assay [2]. diVerent in the two groups. However, graft survival at A positive T-FCXM has been recognized as associ12 and 48 months was significantly poorer in the ated with higher rejections episodes and lower graft positive B-FCXM than in negative B-FCXM (68% vs survival after renal retransplantation [3,4], as well as 90% at 12 months: P=0.007, and 57% vs 79% at 48 after primary renal transplantation [5‐7]. However, in months: P=0.02). Within the positive B-FCXM two recent studies, T-FCXM showed no advantage group, no diVerences were found in pre-transplant over the CDCXM [8,9]. Performing crossmatches on anti-HLA class I or II alloimmunization as well as T and B lymphocytes is justified by the diVerential retransplantation frequency between the patients who expression of HLA class I and II molecules on lympholost their graft and the patients who did not. cytes. In contrast to T lymphocytes, B lymphocytes Conclusion. Our results suggest that a pretransplant express HLA class II molecules constitutively and positive B-FCXM is associated with an impaired long- HLA class I molecules at a higher density than T cells. term graft survival in renal allotransplantation. Positive B-cell crossmatches may be due to antibodies directed against non-HLA antigens (e.g. autoantibod

Laboratoire de Therapeutique Immuno-moleculaire, Etablissement de Transfusion Sanguine de Franche-Comte ´, Service d'Anatomo-pathologie, Service de Radiotherapie-Oncologie, Service d'Hematologie, Centre Hospitalier Universitaire, 25000 Besancon, France, and NCI-FCRDC, Frederick, Maryland Background. We are presently investigating the therapeutic potential of herpes simplex-thymidine ki- nase-expressing donor T cells in the setting of a T cell-depleted allogeneic bone marrow transplantation. The generation, expansion, and selection of the gene- modified T cells require a 12-day ex vivo culture pe- riod in high-dose interleukin (IL)-2 that could signifi- cantly alter their in vivo alloreactivity. Methods. We evaluated the alloreactive potential of such cultured cells in a murine allogeneic bone mar- row transplantation model. Results. The present studies demonstrate that ex vivo-expanded cultured T cells are capable of strong alloreactivity as evidenced by the occurrence of lethal acute graft-versus-host disease (GVHD). However, GVHD mortality after administration of the cultured T cells occurred later than after the administration of a same number of fresh T cells. Similar kinetics of GVHD-induced mortality between cultured and fresh T cells required a 10-fold increase in the number of cultured T cells, indicating a reduced alloreactive po- tential of these cells. The addition of a 2-day "resting" period in low-dose IL-2 resulted in T cells with en- hanced alloreactive potential identical to the alloreac- tivity observed with fresh T cells. Conclusion. Ex vivo IL-2-expanded T cells are capa- ble of significant in vivo alloreactivity. However, an increase in the number of cultured T cells adminis- tered or the introduction of a short resting culture period prior to infusion is necessary in order to achieve in vivo alloreactivity identical to the alloreac- tivity observed with fresh T cells.

International audience; Background: Epstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen.Methods: Peripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool.Results: After immunoselection, a mean of 0.53 ± 0.25 × 106 cells was recovered consisting of a mean of 24.77 ± 18.01% CD4+-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8+-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection.Conclusions: Our results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.

Objective.Screening for latent tuberculosis infection (LTBI) is mandatory before initiating biologics in patients with chronic inflammatory arthritis (CIA). However, few studies have evaluated the discrepancies between the results of tuberculin skin test (TST) and interferon-γ release assays (IGRA) in these patients. The purpose of our study was to investigate factors associated with TST and IGRA results in a large cohort of patients with CIA before the introduction of biologics.Methods.A total of 563 consecutive patients with CIA (293 rheumatoid arthritis, 270 spondyloarthritis) and eligible for biologics were prospectively enrolled. Demographic, clinical, and biological data were recorded. Risk factors for LTBI were assessed. All patients underwent a TST, a chest radiograph, and an IGRA test (T-SPOT.TB).Results.Agreement between the 2 tests was low (κ = 0.16). The bacillus Calmette-Guerin (BCG) status was significantly associated with discordance between the 2 tests (p = 0.004). The TST positivity rate was 34.8%. Factors associated with a negative TST were female sex (p = 0.02) and immunosuppressive treatment (p = 0.003). The only LTBI risk factor associated with TST positivity was an abnormal chest radiograph (p = 0.02). T-SPOT.TB was positive in 21.7% of patients and indeterminate in 15.6%. Previous active TB and chest radiograph abnormalities were associated with IGRA positivity (p = 0.008 and p = 3.9 × 10−5, respectively). The BCG vaccination was associated with negative IGRA (p = 3 × 10−4). Indeterminate IGRA results were associated with age, C-reactive protein, and immunosuppressive treatment (p = 0.005, 0.007, and 0.004, respectively).Conclusion.Our data support the combined use of T-SPOT.TB and TST in patients with CIA before biologics introduction. However, despite these good diagnostic values, indeterminate results may complicate the use of IGRA.

Background. Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Methods. Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells and CD4+ absolute counts. Results. Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08 and 0.18 for CD3%, CD4% CD8% and CD4 absolute counts respectively. Discussion. In-house follow up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 Clinical Cytometry Society.

Melanoma is the most frequent solid tumor associated with leptomeningeal metastasis (LM). The usual diagnostic tools, that is, cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium-enhanced MRI of the entire neuraxis both lack effectiveness. The CellSearch® Veridex technology for the detection of circulating tumor cells (CTC) in blood was designed for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer, which express EpCAM markers. We have previously adapted this technology to detect malignant cells in the CSF of breast cancer LM. Our objective here was to check if this technology would also allow the detection and the enumeration of CTC in the CSF of melanoma patients presenting with LM although melanoma does not express EpCAM markers. On the occasion of the intrathecal treatment of LM in 2 melanoma patients, 5 mL of CSF and 7.5 mL of blood were collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling using a melanoma-dedicated kit. The CellSearch® Veridex technology was then adapted to direct enrichment, enumeration, and visualization of melanoma cells in the CSF. CD146+, HMW-MAA+, CD34−, and CD45− cells with typical morphology could be observed and enumerated sequentially with reproducible results, corresponding to CSF melanoma cells (CSFMC). In contrast to the current gold standard cytomorphological analysis, this new approach allowed a precise quantification of CSFMC in all samples concomitantly analyzed. This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of melanoma patients with LM.

Background The diagnosis of leptomeningeal metastasis (LM) in patients with solid tumors remains difficult. The usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI of the entire neuraxis lack both specificity and sensitivity. The Veridex CellSearch® technology has been designed for the detection of circulating tumor cells (CTC) in blood from cancer patients and validated for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer. Our aim was to adapt this technology for the detection and the enumeration of tumor cells in the CSF of breast cancer patients presenting with LM. Methods On the occasion of a randomized phase III study evaluating the role of the intrathecal treatment in LM from breast cancer (DEPOSEIN, EudraCT N°: 2010-023134-23), the CellSearch® technology was adapted to direct enrichment, enumeration and visualization of tumor cells in 5 mL CSF samples, collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling. Results Sixteen CSF of 8 patients with primary breast cancer presenting with LM were studied. EpCAM+/cytokeratin + cells with typical morphology could be observed and enumerated sequentially with reproducible results in low or elevated numbers in 8 patients. Conclusion This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of cancer patients with LM, especially to appreciate the efficacy of chemotherapy.

Chronic lymphocytic leukemia (CLL), the most common type of leukemia, in spite of recent progress remains incurable. The chronicity of the disease is related at least in part to in vivo resistance ...

Background: The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. Methods: Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10−4. The two best combinations were further applied to 419 samples from 117 treated CLL patients. Results: The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. Conclusions: This manuscript reports on the frequency of CD19+CD5+ B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10−4 white blood cells. © 2011 International Clinical Cytometry Society

Allergic contact dermatitis, the clinical manifestation of contact hypersensitivity, is one of the most common disorders of the skin. It is elicited upon multiple cutaneous re-exposure of sensitized individuals to the sensitizing agent. In this study, we demonstrate that using IL-18 binding protein (IL-18BP) to neutralize IL-18 significantly reduced clinical symptoms in a murine model of contact hypersensitivity. Furthermore, IL-18BP alleviated the relapses during established disease, as indicated by significant protection during re-exposure of mice that had previously undergone a contact hypersensitivity response without treatment. Although edema was not influenced, IL-18BP reduced the number of T cells homing to sites of inflammation, resulting in diminished local production of IFN-γ. Thus, by preventing the accumulation of effector T cells to the target tissue, IL-18BP appears to be a potent protective mediator to counter skin inflammation during contact hypersensitivity. Taken together with the evidence that IL-18 is present in tissue samples of the human disease, our data reinforces IL-18BP as a candidate for this therapeutic indication.