Immunoglobulin G fragment crystallizable glycosylation after hematopoietic stem cell transplantation is dissimilar to donor profiles

Immunoglobulin G (IgG) fragment crystallizable (Fc) N-glycosylation has a large influence on the affinity of the antibody for binding to Fcγ-receptors (FcγRs) and C1q protein, thereby influencing immune effector functions. IgG Fc glycosylation is known to be partly regulated by genetics and partly by stimuli in the microenvironment of the B cell. Following allogeneic hematopoietic stem cell transplantation (HSCT), and in the presence of (almost) complete donor chimerism, IgG is expected to be produced by, and glycosylated in, B cells of donor origin. We investigated to what extent IgG glycosylation in patients after transplantation is determined by factors of the donor (genetics) or the recipient (environment). Using an IgG subclass-specific liquid chromatography-mass spectrometry method, we analyzed the plasma/serum IgG Fc glycosylation profiles of 34 pediatric patients pre-HSCT and at 6 and 12 months post-HSCT and compared these to the profiles of their donors and age-matched healthy controls. Patients treated for hematological malignancies as well as for non-malignant hematological diseases showed after transplantation a lower Fc galactosylation than their donors. Especially for the patients treated for leukemia, the post-HSCT Fc glycosylation profiles were more similar to the pre-HSCT recipient profiles than to profiles of the donors. Pre-HSCT, the leukemia patient group showed as distinctive feature a decrease in sialylation and in hybrid-type glycans as compared to healthy controls, which both normalized after transplantation. Our data suggest that IgG Fc glycosylation in children after HSCT does not directly mimic the donor profile, but is rather determined by persisting environmental factors of the host

Neurophysiological effects of sleep deprivation in healthy adults, a pilot study

Total sleep deprivation (TSD) may induce fatigue, neurocognitive slowing and mood changes, which are partly compensated by stress regulating brain systems, resulting in altered dopamine and cortisol levels in order to stay awake if needed. These systems, however, have never been studied in concert. At baseline, after a regular night of sleep, and the next morning after TSD, 12 healthy subjects performed a semantic affective classification functional magnetic resonance imaging (fMRI) task, followed by a [11C]raclopride positron emission tomography (PET) scan. Saliva cortisol levels were acquired at 7 time points during both days. Affective symptoms were measured using Beck Depression Inventory (BDI), Spielberger State Trait Anxiety Index (STAI) and visual analogue scales. After TSD, perceived energy levels, concentration, and speed of thought decreased significantly, whereas mood did not. During fMRI, response speed decreased for neutral words and positive targets, and accuracy decreased trendwise for neutral words and for positive targets with a negative distracter. Following TSD, processing of positive words was associated with increased left dorsolateral prefrontal activation. Processing of emotional words in general was associated with increased insular activity, whereas contrasting positive vs. negative words showed subthreshold increased activation in the (para)hippocampal area. Cortisol secretion was significantly lower after TSD. Decreased voxel-by-voxel [11 C]raclopride binding potential (BPND) was observed in left caudate. TSD induces widespread cognitive, neurophysiologic and endocrine changes in healthy adults, characterized by reduced cognitive functioning, despite increased regional brain activity

Usage of TCRAV and TCRBV gene families in human fetal and adult TCR rearrangements

We have investigated fetal and adult T-cell receptor (TCR) A and B V-gene repertoires both by fluorescence-activated cell sorter (FACS) analysis with the avialable TCR V region-specific mAbs and by the polymerase chain reaction (PRC) with TRC V gene family-specific oligonucleotides. Among the low number of CD3+ T cells, most of the TRC V region tested for could be detected by FACS analysis in liver, bone marrow, and spleen derived from a 14-week-old fetus and two 15-weeks-old fetuses. Similarly, the PCR analysis showed that the majority of the TCRAV and TCRBV families were expressed in the peripheral organs of the 13-week-old fetus, although an apparent absence of particular TCR V families was found in liver and bone marrow. This was most probably the consequence of the low number of CD3+ T cells in these organs. In 17-week-old week-old fetal thymi the level of expression of some TCRAV and TCRBV gene families, in particular those that contain single member, was lower compared to post-partum thymi and adult peripheral blood mononuclear cells. The combined data of FACS and PCR analysis demonstrate that TCR genes belonging to the majority of TCR V gene families can be used in TCR α and β chain rearrngements during early human fetal life. Our data also suggest that the expression levels of some of the single member TCR V gene families may be influenced by the development stage

Development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay for alemtuzumab quantification in human serum and plasma

Background:Alemtuzumab is a humanized monoclonal antibody that targets the CD52 glycoprotein expressed on most lymphocytes, subsequently inducing complement-mediated and antibody-mediated cytotoxicity. Owing to its ability to induce profound immune depletion, alemtuzumab is frequently used in patients before allogeneic hematopoietic stem cell transplantation to prevent graft rejection and acute graft-versus-host disease. In this clinical context, a stable immunoassay with high sensitivity and specificity to determine alemtuzumab levels is essential for performing pharmacokinetic and pharmacodynamic analyses; however, the available methods have several limitations. Here, we report the successful development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay technique based on commercially available reagents to quantify alemtuzumab in human serum or plasma.Methods:This enzyme-linked immunosorbent assay technique was developed and validated in accordance with the European Medicines Agency guidelines on bioanalytical method validation.Results:The assay sensitivity (lower limit of quantification) is 0.5 ng center dot mL(-1), and the dynamic range is 0.78-25 ng center dot mL(-1). To accommodate quantification of peak concentration and concentrations below the lympholytic level (<0.1 mcg center dot mL(-1)), patients' serum samples were prediluted 20-400 times according to the expected alemtuzumab concentration. The overall within-run accuracy was between 96% and 105%, whereas overall within-run precision (coefficient of variation) was between 3% and 9%. The between-run assessment provided an overall accuracy between 86% and 95% and an overall coefficient of variation between 5% and 14%.Conclusions:The developed assay provides accurate insight into alemtuzumab exposure and its effects on the clinical response to treatment, which is key to optimizing treatment strategies.Personalised Therapeutic

Exposure-response analysis of alemtuzumab in pediatric allogeneic HSCT for nonmalignant diseases: the ARTIC study

Alemtuzumab (anti-CD52 antibody) is frequently prescribed to children with nonmalignant diseases undergoing allogeneic hematopoietic stem cell transplantation (HSCT) to prevent graft failure (GF) and acute graft-versus-host disease (aGVHD). The aim of this multicenter study was the characterization of alemtuzumab population pharmacokinetics to perform a novel modelbased exposure-response analysis in 53 children with nonmalignant immunological or hematological disease and a median age of 4.4 years (interquartile range [IQR], 0.8-8.7). The median cumulative alemtuzumab dose was 0.6 mg/kg (IQR, 0.6-1) administered over 2 to 7 days. A 2-compartment population pharmacokinetics model with parallel linear and nonlinear elimination including allometrically scaled bodyweight (median, 17.50 kg; IQR, 8.76-33.00) and lymphocyte count at baseline (mean, 2.24 x 10(9)/L; standard deviation +/- 1.87) as significant pharmacokinetic predictors was developed using nonlinear mixed effects modeling. Based on the model-estimated median concentration at day of HSCT (0.77 mu g/mL; IQR, 0.33-1.82), patients were grouped into a low- (0.77 mu g/mL) exposure groups. High alemtuzumab exposure at day of HSCT correlated with delayed CD4(+) and CD8(+) T-cell reconstitution (P value = 2, mortality, chimerism at 1 year, viral reactivations, and autoimmunity at a median follow-up of 3.3 years (IQR, 2.5-8.0). In conclusion, this novel population pharmacokinetics model is suitable for individualized intravenous precision dosing to predict alemtuzumab exposure in pediatric allogeneic HSCT for nonmalignant diseases, aiming at the achievement of early T-cell reconstitution and prevention of GF in future prospective studies.Transplantation and immunomodulatio

The frontotemporal syndrome of ALS is associated with poor survival

Thirty percent of ALS patients have a frontotemporal syndrome (FS), defined as behavioral changes or cognitive impairment. Despite previous studies, there are no firm conclusions on the effect of the FS on survival and the use of non-invasive ventilation (NIV) in ALS. We examined the effect of the FS on survival and the start and duration of NIV in ALS. Behavioral changes were defined as >22 points on the ALS-Frontotemporal-Dementia-Questionnaire or ≥3 points on ≥2 items of the Neuropsychiatric Inventory. Cognitive impairment was defined as below the fifth percentile on ≥2 tests of executive function, memory or language. Classic ALS was defined as ALS without the frontotemporal syndrome. We performed survival analyses from symptom ons

A unique immune signature in blood separates therapy-refractory from therapy-responsive acute graft-versus-host disease

Acute graft-versus-host disease (aGVHD) is an immune cell-driven, potentially lethal complication of allogeneic hematopoietic stem cell transplantation affecting diverse organs, including the skin, liver, and gastrointestinal (GI) tract. We applied mass cytometry (CyTOF) to dissect circulating myeloid and lymphoid cells in children with severe (grade III-IV) aGVHD treated with immune suppressive drugs alone (first-line therapy) or in combination with mesenchymal stromal cells (MSCs; second-line therapy). These results were compared with CyTOF data generated in children who underwent transplantation with no aGVHD or age-matched healthy control participants. Onset of aGVHD was associated with the appearance of CD11b(+)CD163(+) myeloid cells in the blood and accumulation in the skin and GI tract. Distinct T-cell populations, including TCR gamma delta(+) cells, expressing activation markers and chemokine receptors guiding homing to the skin and GI tract were found in the same blood samples. CXCR3(+) T cells released inflammation-promoting factors after overnight stimulation. These results indicate that lymphoid and myeloid compartments are triggered at aGVHD onset. Immunoglobulin M (IgM) presumably class switched, plasma-blasts, and 2 distinct CD11b(-) dendritic cell subsets were other prominent immune populations found early during the course of aGVHD in patients refractory to both first- and second-line (MSC-based) therapy. In these nonresponding patients, effector and regulatory T cells with skin- or gut-homing receptors also remained proportionally high over time, whereas their frequencies declined in therapy responders. Our results underscore the additive value of high-dimensional immune cell profiling for clinical response evaluation, which may assist timely decision-making in the management of severe aGVHD.Horizon 2020 (H2020)643580Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Alemtuzumab Induction and Delayed Acute Rejection in Steroid-Free Simultaneous Pancreas-Kidney Transplant Recipients

Transplantation and immunomodulatio

Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome