Aging and End Stage Renal Disease Cause A Decrease in Absolute Circulating Lymphocyte Counts with A Shift to A Memory Profile and Diverge in Treg Population.

There is a growing number of elderly kidney transplant (Ktx) recipients. Elderly recipients present lower acute rejection rates but higher incidence of infection and malignancies. Aging per se seems to result in a shift to memory profile and chronic kidney disease (CKD) in premature immunological aging. Understanding aging and CKD effects on the immune system can improve elderly Ktx immunosuppression. We analyzed the effects of aging and CKD in the immune system, comparing healthy adults (HAd) (n=14, 26±2y), healthy elderly (HEld) (n=15, 79±7y), end stage renal disease (ESRD) adults (EnAd) (n=18, 36±7y) and ESRD elderly (EnEld) (n=31, 65±3y) prior to Ktx regarding their naïve, memory and regulatory T and B peripheral lymphocytes. Aging and ESRD presented additive effect decreasing absolute numbers of B and T-lymphocytes, affecting memory, naive and regulatory subsets without synergic effect. Both resulted in higher percentages of T memory subsets and opposing effects on regulatory T (TREG) subsets, higher percentage in aging and lower in ESRD. Combined effect of aging and ESRD also resulted in higher regulatory B cell percentages. In addition to global lymphopenia and TCD4+ memory shift in both aging and ESRD, aging shifts to an immunoregulatory profile, inducing a increase in TREG percentages, contrasting with ESRD that decreases TREGs. Differential immunosuppression regimens for elderly Ktx may be required. (ClinicalTrials.gov number: NTC01631058).

UPLC-MS/MS assay validation for tacrolimus quantitative determination in peripheral blood T CD4+ and B CD19+ lymphocytes.

Monitoring tacrolimus (Tac) exposure in cell matrices enriched with lymphocytes can improve Tac therapeutic drug monitoring (TDM) in solid organ transplant recipients. An UPLC-MS/MS based assay for Tac quantification in peripheral blood T CD4+ and B CD19+ lymphocytes was developed. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and highly purified (purity >90%) T CD4+ and B CD19+ cell suspensions were acquired by magnetic negative selection from whole blood of 6 healthy volunteers. The purity of lymphocyte suspensions was checked by flow cytometry. Tac extraction was performed in a liquid-liquid zinc sulfate, methanol and acetonitrile based medium. Ascomycin was used as internal standard. The equipment used was a Waters® Acquity™ UPLC system (Waters Corporation, Milford, MA, USA). The chromatographic run was performed on a Waters® MassTrak TDM C18 (2.1 × 10 mm) column (Waters Corporation, Milford, MA, USA). at a flow rate of 0.4 mL/min. The instrument was set in electrospray positive ionization mode. The method was validated according to Clinical Laboratory Standard Institute (CLSI) guidelines and showed a high sensitivity and specificity over a range of 0-5.2 ng/mL in PBMC, 0-5.0 ng/mL in T CD4+ Lymphocytes and 0-5.3 ng/mL in B CD19+ lymphocytes. Precision was appropriate with CV of intra-assay quantifications ranging from 4.9 to 7.4%, and of inter-assay quantifications from 7.2 to 13.9%. Limit of detection and quantification were 0.100 and 0.115 ng/mL in PBMC, 0.058 and 0.109 ng/mL in T CD4+ and 0.017 and 0.150 ng/mL in B CD19+ cells. Matrix effect was not significant among all the studied matrices. Samples showed stability for Tac quantification over a period of 90 days either at room temperature or at -30 °C storage conditions. The method was applied to clinical samples of 20 kidney transplant recipients. Concentrations ranged from 2.200 to 11.900 ng/mL in whole blood, from 0.005 to 0.570 ng/106 cells in PBMC, from 0.081 to 1.432 ng/106 cells in T CD4+, and from 0.197 to 1.564 ng/106 cells in B CD19+ cell matrices. The method has potential applicability for Tac TDM in solid organ transplant recipients.

Bacterial recognition and induced cell activation in sepsis.

The pathogenesis of sepsis involves complex interaction between the host and the infecting microorganism. Recognition and processing of microorganism antigens are essential functions of the cells of innate immune systems, and will ultimately, through the antigen presentation to the cells of adaptive immunity and the synthesis and secretions of mediators, such as cytokines, drive a coordinated immune response. Neutrophils and monocytes will therefore function as sensing and effectors cells. Fundamental in this process is the ability to discriminate self from non-self molecules. Of major interest in sepsis is that the protective and damaging host responses are part of the same process, that is, the inflammatory response that controls the infection process also underscores many of the pathophysiological events of sepsis. Moreover, this is a dynamic process according to the continuum of sepsis and its complications; up and down regulation of cellular activities may be differently regulated in different tissues, different cells and even in different functions of the same cell. This review will focus on microorganism recognition and signalization in sepsis, with emphasis on the neutrophils and monocytes adaptation during the ongoing disease.