Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45+ population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used. © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Inc

The kSORT Assay to Detect Renal Transplant Patients at High Risk for Acute Rejection: Results of the Multicenter AART Study

Development of noninvasive molecular assays to improve disease diagnosis and patient monitoring is a critical need. In renal transplantation, acute rejection (AR) increases the risk for chronic graft injury and failure. Noninvasive diagnostic assays to improve current late and nonspecific diagnosis of rejection are needed. We sought to develop a test using a simple blood gene expression assay to detect patients at high risk for AR. We developed a novel correlation-based algorithm by step-wise analysis of gene expression data in 558 blood samples from 436 renal transplant patients collected across eight transplant centers in the US, Mexico, and Spain between 5 February 2005 and 15 December 2012 in the Assessment of Acute Rejection in Renal Transplantation (AART) study. Gene expression was assessed by quantitative real-time PCR (QPCR) in one center. A 17-gene set—the Kidney Solid Organ Response Test (kSORT)—was selected in 143 samples for AR classification using discriminant analysis (area under the receiver operating characteristic curve [AUC] = 0.94; 95% CI 0.91–0.98), validated in 124 independent samples (AUC = 0.95; 95% CI 0.88–1.0) and evaluated for AR prediction in 191 serial samples, where it predicted AR up to 3 mo prior to detection by the current gold standard (biopsy). A novel reference-based algorithm (using 13 12-gene models) was developed in 100 independent samples to provide a numerical AR risk score, to classify patients as high risk versus low risk for AR. kSORT was able to detect AR in blood independent of age, time post-transplantation, and sample source without additional data normalization; AUC = 0.93 (95% CI 0.86–0.99). Further validation of kSORT is planned in prospective clinical observational and interventional trials. The kSORT blood QPCR assay is a noninvasive tool to detect high risk of AR of renal transplants

Fcγ Receptors in Solid Organ Transplantation.

In the current era, one of the major factors limiting graft survival is chronic antibody-mediated rejection (ABMR), whilst patient survival is impacted by the effects of immunosuppression on susceptibility to infection, malignancy and atherosclerosis. IgG antibodies play a role in all of these processes, and many of their cellular effects are mediated by Fc gamma receptors (FcγRs). These surface receptors are expressed by most immune cells, including B cells, natural killer cells, dendritic cells and macrophages. Genetic variation in FCGR genes is likely to affect susceptibility to ABMR and to modulate the physiological functions of IgG. In this review, we discuss the potential role played by FcγRs in determining outcomes in solid organ transplantation, and how genetic polymorphisms in these receptors may contribute to variations in transplant outcome.MRC is supported by the NIHR Cambridge BRC, the NIHR Blood and Transplant Research Unit (Cambridge) and by a Medical Research Council New Investigator Grant (MR/N024907/1).This is the final version of the article. It first appeared from Springer via https://doi.org/10.1007/s40472-016-0116-

Alternative low-cost adsorbent for water and wastewater decontamination derived from eggshellwaste: an overview

As the current global trend towards more stringent environmental standards, technical applicability and cost-effectiveness became key factors in the selection of adsorbents for water and wastewater treatment. Recently, various low-cost adsorbents derived from agricultural waste, industrial by-products or natural materials, have been intensively investigated. In this respect, the eggshells from egg-breaking operations constitute significant waste disposal problems for the food industry, so the development of value-added by-products from this waste is to be welcomed. The egg processing industry is very competitive, with low profit margins due to global competition and cheap imports. Additionally, the costs associated with the egg shell disposal (mainly on landfill sites) are significant, and expected to continue increasing as landfill taxes increase. The aim of the present review is to provide an overview on the development of low-cost adsorbents derived from eggshell by-products

Human Cytomegalovirus Fcγ Binding Proteins gp34 and gp68 Antagonize Fcγ Receptors I, II and III

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.DFG grant He 2526/6-2.European Commission grants QLRT-2001-01112 and MRTN-CT-2005-019248.Helmholtz Association through VISTRIE VH-VI-242.UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose tissue

Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose tissue. Obes Res. 2005;13: 974-981. Objective: Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)-adenosine pathway in adipose tissue. Research Methods and Procedures: Sixteen miniature Yucatan swine (Sits scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 mu L/min with Ringer\u27s solution containing no addition, varying levels of cyclic AMP, 10 mu M isoproterenol, or 10 mu M isoproterenol plus 1 mM alpha,beta-methylene adenosine 5\u27-diphosphate (AMPCP), a 5\u27-nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 mu M isoproterenol, or 1 mu M isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Results: Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP-provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. Discussion: These data suggest the existence of a cyclic AMP-adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified