Mechanisms of action of antithymocyte globulin: old dogs with new tricks!

International audienceUnderstanding the mechanisms underlying immunological tolerance is the key to successful transplantation. Recent findings demonstrate that polyclonal antibodies such as the antithymocyte globulins preparations can provide wide spectrum immunomodulation, suggesting that their use in the immunosuppression therapeutic armamentarium may help in reducing the incidence of organ rejection and improving patients' outcome after hematopoietic stem cell transplantation

Development and validation of an LC-MS/MS method for the analysis of ivermectin in plasma, whole blood, and dried blood spots using a fully automatic extraction system.

Ivermectin is deployed in mass drug administration (MDA) campaigns to control parasitic diseases in the tropics, with billions of treatments having been administered in the last three decades. Simple blood sampling tools, like the dried blood spots (DBS) technique, are needed to monitor treatments in such challenging settings. Thus, we developed a fully automated method for the analysis of ivermectin in DBS microsamples, including a bioanalytical and clinical validation. Automated extraction was carried out using a DBS-MS 500 autosampler which was coupled to a LC-MS/MS system. DBS were extracted with 20 μL solvent and eluted on a C8 analytical column. Analysis was performed by multiple reaction monitoring in the positive mode. Automated DBS extraction resulted in consistent recoveries (62.8 ± 4.3%) and matrix effects (68.0 ± 8.1%) between different donors and concentration levels. Intra- and inter-day accuracy and precision deviations were ≤15%, while samples with hematocrits from 20 to 60% could be quantified reliably. The achieved sensitivity of 1 ng/mL in DBS samples is sufficient to analyze ivermectin at the dose given (single oral administration of 12 mg) over a period of at least 72 h post treatment. Importantly, DBS samples are stable after one-month storage at room temperature (accuracy: 88.8-96.2%), thus samples collected in the field must not be shipped on dry ice. Ivermectin concentrations in venous and capillary blood agreed strongly, with a mean difference of -4.8%. Moreover, the drying process of DBS did not alter the analysis and importantly plasma concentrations can be estimated from DBS data using the hematocrit and red blood cell partitioning as correction factor. Our method enables uncomplicated sample collection and shipment as well as automated analysis of large amounts of samples, which is key to surveying MDA campaigns in remote settings

Involvement of the CX3CL1 (fractalkine)/ CX3CR1 pathway in the pathogenesis of acute graft-versus-host disease

International audienceThis study investigated the role of cytokines and chemokines in aGVHD incidence and severity in 109 patients who underwent reduced-intensity conditioning allogeneic stem cell transplantation (HSCT). Among the 42 cytokines tested at d 0 HSCT, only CX3CL1 levels at d 0 HSCT were significantly associated with Grades II–IV aGVHD development (P = 0.04). Increased levels of CX3CL1 at d 20–30 and 50 post-HSCT were also significantly associated with aGVHD (P = 0.02 and P = 0.03, respectively). No such association was found before the conditioning regimen or at d 100–120 post-HSCT. As the receptor for CX3CL1 is CX3CR1, the number of CX3CR1 + cells was determined by flow cytometry. The CX3CR1 + CD8 + T cell proportion was significantly higher in patients with aGVHD than those without aGVHD (P = 0.01). To investigate the distribution of the CX3CL1/CX3CR1 axis in the anatomic sites of aGVHD, CX3CL1 and CX3CR1 levels were studied by use of an in situ immunohistochemical analysis on GI biopsies of patients with intestinal aGVHD. CX3CL1 expression was increased significantly in the epithelial cells and mononuclear cells of the lamina propria. CX3CR1 + mononuclear cells were identified in close contact with epithelial cells. These findings strongly suggest the implication of the CX3CL1/CX3CR1 axis in the pathogenesis of aGVHD. J. Leukoc. Biol. 97: 227–235; 2015

Analysis of spontaneous tumor-specific CD4 T-cell immunity in lung cancer using promiscuous HLA-DR telomerase-derived epitopes: potential synergistic effect with chemotherapy response.

International audiencePURPOSE: To investigate the presence and impact of spontaneous telomerase-specific CD4 T-cell responses in cancer patients. EXPERIMENTAL DESIGN: A multistep approach was used to design novel pan-HLA-DR-restricted peptides from telomerase. T-cell clones isolated from cancer patients were used to characterize the polarization of telomerase-specific CD4 response. The presence of spontaneous CD4 T-cell response against telomerase was monitored in 84 metastatic non-small cell lung cancer (NSCLC) patients before first-line chemotherapy (CT) using IFN-γ ELISPOT assay. Then we analyzed the impact of the pretherapeutic telomerase-specific CD4 T immunity on clinical outcome in patients according to their respective response to CT. RESULTS: We described four novel telomerase-derived CD4 epitopes referred as universal cancer peptides (UCP) that effectively bind to most commonly found human MHC class II alleles. UCP-specific CD4 T-cell repertoire is present in human and UCP-specific CD4 T-cell clones generated from cancer patients exhibited high avidity and are Th1 polarized. Significant frequency (38%) of naturally occurring UCP-specific T-cell responses were detected before CT in advanced NSCLC but not in healthy volunteers. This response was shown to significantly increase overall survival (OS) of patients responding to CT (Median OS: 53 vs. 40 weeks, P = 0.034). CONCLUSIONS: These results show for the first time a potential synergistic effect of telomerase-specific CD4 T-cell response with CT response in NSCLC and underline the potential role of tumor-specific CD4 T-cell response on the efficiency of conventional anticancer therapy

Increased regulatory T-cell numbers are associated with farm milk exposure and lower atopic sensitization and asthma in childhood

European cross-sectional studies have suggested that prenatal and postnatal farm exposure decreases the risk of allergic diseases in childhood. Underlying immunologic mechanisms are still not understood but might be modulated by immune-regulatory cells early in life, such as regulatory T (Treg) cells.; We sought to assess whether Treg cells from 4.5-year-old children from the Protection against Allergy: Study in Rural Environments birth cohort study are critical in the atopy and asthma-protective effect of farm exposure and which specific exposures might be relevant.; From 1133 children, 298 children were included in this study (149 farm and 149 reference children). Detailed questionnaires until 4 years of age assessed farming exposures over time. Treg cells were characterized as upper 20% CD4(+)CD25(+) forkhead box protein 3 (FOXP3)(+) (intracellular) in PBMCs before and after stimulation (with phorbol 12-myristate 13-acetate/ionomycin or LPS), and FOXP3 demethylation was assessed. Atopic sensitization was defined by specific IgE measurements; asthma was defined by a doctor's diagnosis.; Treg cells were significantly increased in farm-exposed children after phorbol 12-myristate 13-acetate/ionomycin and LPS stimulation. Exposure to farm milk was defined as a relevant independent farm-related exposure supported by higher FOXP3 demethylation. Treg cell (upper 20% CD4(+)CD25(+), FOXP3(+) T cells) numbers were significantly negatively associated with doctor-diagnosed asthma (LPS stimulated: adjusted odds ratio, 0.26; 95% CI, 0.08-0.88) and perennial IgE (unstimulated: adjusted odds ratio, 0.21; 95% CI, 0.08-0.59). Protection against asthma by farm milk exposure was partially mediated by Treg cells.; Farm milk exposure was associated with increased Treg cell numbers on stimulation in 4.5-year-old children and might induce a regulatory phenotype early in life, potentially contributing to a protective effect for the development of childhood allergic diseases