In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+ cells mature in the thymus

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(c)(-/-) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alphabeta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT

Gene Set Enrichment Analysis Identifies LIF as a Negative Regulator of Human Th2 Cell Differentiation

In this study we show that IL-4 is crucial during reinforcement window of human Th2 differentiation for optimal Th2 development. We have also shown here that during this stage, IL-4 helps in cellular decision-making process of differentiation versus proliferation. We have combined computational and experimental methods to analyze Th2 transcription network to name novel players of the process of Th2 differentiation. Here we report that LIF through STAT3 negatively regulates Th2 differentiation. This approach can be generalized to analyze “omics” data to identify key regulatory modules

T cells fail to develop in the human skin-cell explants system; an inconvenient truth

BACKGROUND: Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system. RESULTS: Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes. CONCLUSIONS: Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future

The prevalence of antibodies against the HLA-DRB3 protein in kidney transplantation and the correlation with HLA expression

Human leukocyte antigen (HLA)-DRB3 is a functional HLA class II gene, which has a limited allele diversity in the human population. Furthermore, the HLA-DRB3 gene is only present in a subset of individuals. Therefore, in organ transplantation, this HLA molecule is frequently mismatched between patient and graft donor and thus antibodies against this mismatched HLA molecule can develop. In this study, we aimed to evaluate the prevalence and reactivity of these antibodies and aimed to identify factors that underlie antibody formation against HLA-DRB3. We showed in our patient cohort that HLA-DRB3 antibodies are identified in about 7% of all patients that were screened with solid phase assays. In these assays, we observed multiple antibody reactivity patterns indicating that HLA-DRB3 harbours multiple epitopes. In those cases, where we succeeded at tracing back the induction of these antibodies to the molecular HLA typing of the immunogenic event, we noticed a different frequency of HLA-DRB1 allele groups in the donors as compared to a control group. To a certain extent this distribution (e.g. HLA-DRB1* 11 individuals) could be linked to an altered expression level. However, it also appears that different HLA-DRB3 alleles (e.g. HLA-DRB3* 01 group) vary in their immunogenicity without having an expression difference. In conclusion, our study provides information on the immunogenicity and reactivity patterns of antibodies against HLA-DRB3 in kidney transplantation, and it points towards the possibility of HLA expression as a factor underlying antibody formation

PIRCHE-II Is Related to Graft Failure after Kidney Transplantation

Individual HLA mismatches may differentially impact graft survival after kidney transplantation. Therefore, there is a need for a reliable tool to define permissible HLA mismatches in kidney transplantation. We previously demonstrated that donor-derived Predicted Indirectly ReCognizable HLA Epitopes presented by recipient HLA class II (PIRCHE-II) play a role in de novo donor-specific HLA antibodies formation after kidney transplantation. In the present Dutch multi-center study, we evaluated the possible association between PIRCHE-II and kidney graft failure in 2,918 donor–recipient couples that were transplanted between 1995 and 2005. For these donors–recipients couples, PIRCHE-II numbers were related to graft survival in univariate and multivariable analyses. Adjusted for confounders, the natural logarithm of PIRCHE-II was associated with a higher risk for graft failure [hazard ratio (HR): 1.13, 95% CI: 1.04–1.23, p = 0.003]. When analyzing a subgroup of patients who had their first transplantation, the HR of graft failure for ln(PIRCHE-II) was higher compared with the overall cohort (HR: 1.22, 95% CI: 1.10–1.34, p < 0.001). PIRCHE-II demonstrated both early and late effects on graft failure in this subgroup. These data suggest that the PIRCHE-II may impact graft survival after kidney transplantation. Inclusion of PIRCHE-II in donor-selection criteria may eventually lead to an improved kidney graft survival

Evaluation of spelt germplasm for polyphenol oxidase activity and aluminium resistance

Kidney transplantation is the best treatment option for patients with end-stage renal failure. At present, approximately 800 Dutch patients are registered on the active waiting list of Eurotransplant. The waiting time in the Netherlands for a kidney from a deceased donor is on average between 3 and 4years. During this period, patients are fully dependent on dialysis, which replaces only partly the renal function, whereas the quality of life is limited. Mortality among patients on the waiting list is high. In order to increase the number of kidney donors, several initiatives have been undertaken by the Dutch Kidney Foundation including national calls for donor registration and providing information on organ donation and kidney transplantation. The aim of the national PROCARE consortium is to develop improved matching algorithms that will lead to a prolonged survival of transplanted donor kidneys and a reduced HLA immunization. The latter will positively affect the waiting time for a retransplantation. The present algorithm for allocation is among others based on matching for HLA antigens, which were originally defined by antibodies using serological typing techniques. However, several studies suggest that this algorithm needs adaptation and that other immune parameters which are currently not included may assist in improving graft survival rates. We will employ a multicenter-based evaluation on 5429 patients transplanted between 1995 and 2005 in the Netherlands. The association between key clinical endpoints and selected laboratory defined parameters will be examined, including Luminex-defined HLA antibody specificities, T and B cell epitopes recognized on the mismatched HLA antigens, non-HLA antibodies, and also polymorphisms in complement and Fc receptors functionally associated with effector functions of anti-graft antibodies. From these data, key parameters determining the success of kidney transplantation will be identified which will lead to the identification of additional parameters to be included in future matching algorithms aiming to extend survival of transplanted kidneys and to diminish HLA immunization. Computer simulation studies will reveal the number of patients having a direct benefit from improved matching, the effect on shortening of the waiting list, and the decrease in waiting time

PIRCHE-II is related to graft failure after kidney transplantation

Individual HLA mismatches may differentially impact graft survival after kidney transplantation. Therefore, there is a need for a reliable tool to define permissible HLA mismatches in kidney transplantation. We previously demonstrated that donor-derived Predicted Indirectly ReCognizable HLA Epitopes presented by recipient HLA class II (PIRCHE-II) play a role in de novo donor-specific HLA antibodies formation after kidney transplantation. In the present Dutch multi-center study, we evaluated the possible association between PIRCHE-II and kidney graft failure in 2,918 donor-recipient couples that were transplanted between 1995 and 2005. For these donors-recipients couples, PIRCHE-II numbers were related to graft survival in univariate and multivariable analyses. Adjusted for confounders, the natural logarithm of PIRCHE-II was associated with a higher risk for graft failure [hazard ratio (HR): 1.13, 95% CI: 1.04-1.23, p = 0.003]. When analyzing a subgroup of patients who had their first transplantation, the HR of graft failure for ln(PIRCHE-II) was higher compared with the overall cohort (HR: 1.22, 95% CI: 1.10-1.34, p < 0.001). PIRCHE-II demonstrated both early and late effects on graft failure in this subgroup. These data suggest that the PIRCHE-II may impact graft survival after kidney transplantation. Inclusion of PIRCHE-II in donor-selection criteria may eventually lead to an improved kidney graft survival

Detection of Anti-neutrophil Cytoplasmic Antibodies (ANCA) by Indirect Immunofluorescence

The eventual presence of anti-neutrophil cytoplasmic antibodies (ANCA) can initially be screened with indirect immunofluorescence (IIF). The majority of laboratories that facilitate ANCA testing use commercial kits. Although in-house assays are not encouraged in routine clinical laboratories, knowledge on the methodological aspects of the assay remains of importance. These aspects include choice of substrate, choice of fixative, staining procedure, and interpretation procedure. In this paper details on the methodology are provided and discussed in the context of the clinical application

Antigen-Specific Detection of Autoantibodies Against Myeloperoxidase (MPO) and Proteinase 3 (PR3)

ANCA testing was introduced in many laboratories throughout the world when it was recognized that a significant subset of patients with small vessel vasculopathies presented with such antibodies. Many laboratories developed and introduced in-house testing methods for antigen-specific ANCA detection complementary to indirect immune fluorescence screening. Such in-house tests have proven their merit in diagnosing vasculitis and were important to identify critical steps in the development of antigen-specific assays with high sensitivity and specificity. In the meantime various commercial assays became available for antigen-specific ANCA testing. Because of the high diagnostic accuracy of such assays it can be anticipated that commercial, antigen-specific tests will completely replace in-house testing for MPO- and PR3-ANCA. Furthermore, such tests will replace the need for IIF in the diagnostic workup of AAV. In this light it can be foreseen that the knowledge that underlies the development of in-house ANCA testing will gradually disseminate over time. Therefore we describe the current antigen-specific ANCA ELISAs (direct and capture) with the intention to maintain the knowledge and the identification of the critical steps in the development of robust assays

Assessing the feasibility and effectiveness of household-pooled universal testing to control COVID-19 epidemics.

Outbreaks of SARS-CoV-2 are threatening the health care systems of several countries around the world. The initial control of SARS-CoV-2 epidemics relied on non-pharmaceutical interventions, such as social distancing, teleworking, mouth masks and contact tracing. However, as pre-symptomatic transmission remains an important driver of the epidemic, contact tracing efforts struggle to fully control SARS-CoV-2 epidemics. Therefore, in this work, we investigate to what extent the use of universal testing, i.e., an approach in which we screen the entire population, can be utilized to mitigate this epidemic. To this end, we rely on PCR test pooling of individuals that belong to the same households, to allow for a universal testing procedure that is feasible with the limited testing capacity. We evaluate two isolation strategies: on the one hand pool isolation, where we isolate all individuals that belong to a positive PCR test pool, and on the other hand individual isolation, where we determine which of the individuals that belong to the positive PCR pool are positive, through an additional testing step. We evaluate this universal testing approach in the STRIDE individual-based epidemiological model in the context of the Belgian COVID-19 epidemic. As the organisation of universal testing will be challenging, we discuss the different aspects related to sample extraction and PCR testing, to demonstrate the feasibility of universal testing when a decentralized testing approach is used. We show through simulation, that weekly universal testing is able to control the epidemic, even when many of the contact reductions are relieved. Finally, our model shows that the use of universal testing in combination with stringent contact reductions could be considered as a strategy to eradicate the virus