Point-of-care tests are highly valuable in providing fast results for medical decisions for greater flexibility in patient care. Many diagnostic tests, such as ELISAs, that are commonly used within ...clinical laboratory settings require trained technicians, laborious workflows, and complex instrumentation hindering their translation into point-of-care applications. Herein, we demonstrate the use of a homogeneous, bioluminescent-based, split reporter platform that enables a simple, sensitive, and rapid method for analyte detection in clinical samples. We developed this point-of-care application using an optimized ternary, split-NanoLuc luciferase reporter system that consists of two small reporter peptides added as appendages to analyte-specific affinity reagents. A bright, stable bioluminescent signal is generated as the affinity reagents bind to the analyte, allowing for proximity-induced complementation between the two reporter peptides and the polypeptide protein, in addition to the furimazine substrate. Through lyophilization of the stabilized reporter system with the formulated substrate, we demonstrate a shelf-stable, all-in-one, add-and-read analyte-detection system for use in complex sample matrices at the point-of-care. We highlight the modularity of this platform using two distinct SARS-CoV-2 model systems: SARS-CoV-2 N-antigen detection for active infections and anti-SARS-CoV-2 antibodies for immunity status detection using chemically conjugated or genetically fused affinity reagents, respectively. This technology provides a simple and standardized method to develop rapid, robust, and sensitive analyte-detection assays with flexible assay formatting making this an ideal platform for research, clinical laboratory, as well as point-of-care applications utilizing a simple handheld luminometer.
Detection of posttransplant donor‐specific anti‐HLA antibodies (DSA) constitutes a risk factor for kidney allograft loss. Together with complement activation, NK‐cell antibody‐dependent cell mediated ...cytotoxicity (ADCC) has been proposed to contribute to the microvascular damage associated to humoral rejection. In the present observational exploratory study, we have tried to find a relationship of circulating donor‐specific and nondonor‐specific anti‐HLA antibodies (DSA and HLA non‐DSA) with peripheral blood NK‐cell subsets and clinical features in 393 renal allograft recipients. Multivariate analysis indicated that retransplantation and pretransplant sensitization were associated with detection of posttransplant DSA. Recipient female gender, DR mismatch and acute rejection were significantly associated with posttransplant DSA compared to HLA non‐DSA. In contrast with patients without detectable anti‐HLA antibodies, DSA and HLA non‐DSA patients displayed lower proportions of NK‐cells, associated with increased CD56bright and NKG2A+ subsets, the latter being more marked in DSA cases. These differences appeared unrelated to retransplantation, previous acute rejection or immunosuppressive therapy. Although preliminary and observational in nature, our results suggest that the assessment of the NK‐cell immunophenotype may contribute to define signatures of alloreactive humoral responses in renal allograft recipients.
This single‐center prospective study of kidney transplant recipients shows that patients with detectable anti‐HLA antibodies display lower proportions of NK cells and increased CD56bright and NKG2A+ subsets than patients without antibodies, the latter being more marked in cases with donor‐specific antibodies.
We have recently reported that some lymphocyte populations do not maintain the same proportion in kidney graft blood as in peripheral blood, despite a stable function of the transplanted kidney. ...These results suggest that a comparative study between leukocyte cells from graft blood and those obtained from peripheral blood could provide information about the inflammatory state of the transplanted organ. In this work we selected the population of CD4+ lymphocytes and monocytes expressing CXCR3 to test this hypothesis.
The study was performed by flow cytometry during month 3, 6, and 12 after transplantation in 58 patients who received an isolated kidney transplant and the same immunosuppressive regimen. The peripheral blood sample was obtained by venipuncture and the graft blood by fine needle aspiration.
We found a significant percentage decrease in CXCR3+ monocytes throughout the first year of transplantation in peripheral blood (15.9 ± 20.7 vs. 12.6 ± 12.4 vs. 6.3 ± 9.0, at 3, 6, and 12 months, respectively; P = .001), whereas the percentage of CXCR3+ monocytes in graft blood did not change over this period. This situation resulted in a significant percentage difference between the CXCR3+ monocytes from the graft blood and those from the peripheral blood at the sixth (15.8 ± 8.1 vs. 12.6 ± 12.4, respectively; P = .008) and 12th months (12.9 ± 8.1 vs. 6.3 ± 9.0, respectively; P < .001).
Therefore, we can conclude that the significant percentage increase of CXCR3+ monocytes in graft blood with respect to peripheral blood suggests the presence of inflammatory activity despite renal function being stable during the second half of the first year post-transplantation.
Abstract Background The Cylex Immuknow assay provides a rapid assessment of global immune function in immunocompromised patients by measuring the global immune responses of CD4 T cells from a ...whole-blood sample. It may help to monitor the immune status of immunosuppressed transplant patients. However, earlier studies have shown that there is no consensus on the utility of the Immuknow assay in renal transplant rejection. Methods T-cell activation was determined by measuring an increase of intracellular adenosine triphosphate (iATP) from CD4 cells in 227 samples from 116 kidney transplant patients. The results were analyzed regarding patient clinical status, namely, rejection, infection, or stability. In addition, we measured the immunologic response of 108 healthy control subjects. Results There were 24 infectious and 36 rejection episodes. iATP concentrations differed significantly between stable and infected patients (180.5 ± 55.2 vs 375.3 ± 140.1 ng/mL; P < .001) and between infected patients and control subjects (180.5 ± 55.2 vs 436.5 ± 112 ng/mL; P < .001). No correlation was observed between patients suffering an acute rejection episode with this response. Conclusions Our results confirmed that the Immuknow assay identified transplant patients at risk for infection. It may provide information to guide immunosuppressive therapy, but the assay did not seem to have the potential to differentiate subjects experiencing rejection.
Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies ...directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.
Abstract Background The cytochrome P450 3A5 (CYP3A5) enzyme has been implicated to determine blood pressure (BP) in humans. Different results have been reported concerning CYP3A5 gene polymorphisms ...and posttransplantation hypertension in kidney recipients. Our objective was to investigate whether CYP3A5*1/*3 polymorphism was associated with ambulatory BP among a population of renal transplant recipients receiving the calcineurin inhibitor tacrolimus for immunosuppression. Methods Sixty primary kidney transplant recipients undergoing treatment with tacrolimus were genotyped for the CYP3A5*1/*3 polymorphism. We analysed the association of the CYP3A5 alleles with ambulatory systolic and diastolic BP measured at 6 and 24 months posttransplantation. Results We observed that 23.3% of the patients were CYP3A5*1 carriers and 76.7% were homozygous for CYP3A5*3. CYP3A5*1 carriers showed higher adjusted systolic BP and diastolic BP at 6 and 24 months posttransplantation, and they were prescribed more antihypertensive drugs compared with non CYP3A5*1 carrier patients, albeit not significant. No significant differences were found comparing the distribution of the hypertension classes. Conclusion We did not observe a significant association of CYP3A5*1/*3 polymorphism with posttransplantation hypertension, although there were some differences in BP associated with the presence of the CYP3A5*1 allele.
In haploidentical stem cell transplantation (SCT), the “ideal donor” selection is not performed in a standardized way according to killer cell immunoglobulin–like receptor (KIR) genotype expressed by ...potential donors. The aim of this study was to evaluate the relevance of KIR genotype in a series of patients submitted to haploidentical SCT in our center.
We retrospectively analyzed 30 patients that were prepared with the use of a conditioning regimen including thiotepa-busulfan-fludarabine with high doses of post-transplantation cyclophosphamide (CyPT) and tacrolimus as graft-versus-host disease (GVHD) prophylaxis. We analyzed the impact of the KIR genotype variables (donor AA/Bx haplotype, donor B content, KIR inhibitor mismatches, and mismatching in KIR ligands in the graft-versus-host direction and the host-versus-graft direction) on overall survival, GVHD-free survival, and event-free survival.
Statistical significance was found for the presence of mismatches on KIR ligands in the graft-versus-host direction in relation to the diagnosis of chronic GVHD (54% vs 100%; P = .004). Significance was also found for the effect of the donor presence AA or Bx haplotype in relation to the diagnosis of chronic GVHD (50% vs 86%; P = .033).
KIR genotyping can provide useful information that can help us with the right donor choice for haploidentical SCT without T-cell depletion with high doses of CyPT. Donors with Bx haplotype that do not show KIR ligand incompatibilities in the graft-versus-host direction may provide a lower risk of GVHD.
Abstract An important factor affecting the success in the setting of related haploidentical hematopoietic stem cell transplantation (HSCT) is the graft-versus-leukemia effect mediated by natural ...killer (NK) cells when the donor displays NK alloreactivity versus the recipient. NK cell function is regulated by killer immunoglobulin-like receptors (KIR) and it has been described that donor KIR genotype influences transplantation outcome. This has led to a requirement of laboratories to have a quality assurance program for validation and control of their KIR genotyping methods. The goal of the 1st and 2nd Spanish KIR Genotyping Workshops was to provide an external proficiency testing program in KIR genotyping for Spanish immunology and transplant laboratories. These workshops were conducted during the years 2014–2016 and consisted of 17 participating laboratories typing a set of 20 samples. The presence/absence of 16 mandatory KIR loci (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 2DP1, 3DL1, 3DL2, 3DL3, 3DS1, and 3DP1) was evaluated per sample. Methods for KIR genotyping included polymerase chain reaction with the use of sequence-specific primers and sequence-specific oligoprobes. Consensus typing was reached in all samples, and the performance of laboratories in external proficiency testing was satisfactory in all cases. The polymorphism detected in the small sample studied in both workshops is indicative of an ample variety of KIR gene profiles in the Spanish population.
Human leukocyte antigen (HLA) study in Murcian individuals was performed in order to provide information of their historical origins and relationships with other Iberian and Mediterranean ...populations. HLA class I and class II alleles were determined in 173 unrelated Caucasoid donors from Murcia Region in the Southeast of Spain by serologic and DNA based polymerase chain reaction (PCR) typing. Class I antigen and class II allele frequencies of our series were not very different to those found in Spaniards. The analysis of extended haplotypes showed that the three haplotypes most frequent in our population were respectively, A29-B44-Cwb-DRB1*0701-DRB4*0101-DQA1*0201-DQB1*0202, A1-B8-Cw7-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201 and A30-B18-Cw5-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201. They were followed by A26-B38-Cwb-DRB1*1301-DRB3*0202-DQA1*0103-DQB1*0603, which could point to an ancestral relationship between Murcian and Portuguese Iberian populations, and by A2-B7-Cw7-DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 also present in all Iberian Peninsula populations. Allelic frequencies, populations distance dendrogram and correspondence analysis were used to study the relationships between Murcian and other populations. The closest relation was observed with Spaniards and Portuguese, followed in decreasing order by French, Italians, Algerians, Germans, Catalans, Basques, Cretans, Sardinians, and Greeks. Thus, Murcian population seems to belong to the European genetic pool, revealing a lesser genetic distance with the North Africans and the rest of populations from the Iberian Peninsula.
The ever increasing price pressure on the worldwide launch market has shaped the strategy for the next generation of European launchers preparation. In addition to the development of low-cost ...propulsion technologies based on LOX/CH4, CNES Launchers Directorate, investigate the introduction of reuse of the main stage with a return phase performed by a Toss-Back maneuver, reducing overall costs.
These launcher concepts, which have never been operated from French Guiana, give rise to new safety concerns different from those already handled for the launches operated from GSC, challenging the flight safety principles.
This article provides an overview of the most critical scenarios, following an accidental explosion, in a Toss-Back maneuver, with an analysis of the effects of the resulting explosion (in terms of energy and fragmentation), and its impact on ground. The main goal is to identify the most critical parameters driving the scenarios’ criticality and to determine the possible risk mitigation measures such as “design-to-safety” solutions.
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