Sensitivity of activated human lymphocytes to cyclosporine and its metabolites

Alloreactive T cells generated as clones from mixed lymphocyte cultures, or propagated from heart or liver transplant biopsies, were tested for secondary proliferation measured in the primed lymphocyte test in the presence of Cyclosporine A and metabolites fractionated from human bile. Significant differences were observed in Cyclosporine A sensitivity between various cell cultures ranging as high as 100-fold. The liver is the primary site of Cyclosporine A metabolism, which yields a number of hydroxylated and N-dimethylated derivatives that are eventually secreted into the bile. Bile was collected from adult liver transplant patients on Cyclosporine A therapy and following extraction with diethyl ether, separated by high pressure liquid chromatography. Thirteen fractions were tested for their effect on lymphocyte proliferation in concanavalin A activation, mixed lymphocyte cultures and primed lymphocyte test assays. The strongest immunosuppressive effect was found with fraction 8, which contained metabolite M17, which has a single hydroxylation in position 1. Only three other fractions 9, 10, and 13, which contained metabolites M1, M18, and M21, respectively, exhibited immunosuppressive activity, albeit much lower than that of Cyclosporine A. Differences in Cyclosporine A sensitivity among alloreactive T cells followed similar patterns with Cyclosporius A metabolites. Thus, the assessment of the Cyclosporine A effect must consider differences in drug sensitivity of lymphocytes involved in transplant immunity and the generation of metabolites with immunosuppressive activity. © 1988

Analysis of chronic rejection and obliterative arteriopathy: Possible contributions of donor antigen-presenting cells and lymphatic disruption

Sequential analysis of changes that lead to chronic rejection was undertaken in an animal model of chronic rejection and obliterative arteriopathy. Brown Norway rats are pretreated with a Lewis bone marrow infusion or a Lewis orthotopic liver allograft and a short course of immunosuppression. They are challenged 100 days later with a Lewis heterotopic heart graft without immunosuppression. The heart grafts in both groups undergo a transient acute rejection, but all rats are operationally tolerant; the heart grafts are accepted and remain beating for more than 100 days. Early arterial remodeling, marked by arterial bromodeoxyuridine incorporation, occurred in both groups between 5 and 30 days during the transient acute rejection. It coincided with the presence of interstitial (but not arterial intimal) inflammation and lymphatic disruption and resulted in mild intimal thickening. Significant arterial narrowing occurred only in the bone-marrow-pretreated rats between 60 and 100 days. It was associated with T lymphocyte and macrophage inflammation of the heart graft that accumulated in the endocardium and arterial intima and adventitia near draining lymphatics. There also was loss of passenger leukocytes from the heart graft, up-regulation of cytokine mRNA and major histocompatibility class II on the endothelium, and focal disruption of lymphatics. In contrast, long-surviving heart grafts from the Lewis orthotopic liver allograft pretreated group are near normal and freedom from chronic rejection in this group was associated with persistence of donor major histocompatibility class-II-positive hematolymphoid cells, including OX62+ donor dendritic cells. This study offers insights into two different aspects of chronic rejection: 1) possible mechanisms underlying the persistent immunological injury and 2) the association between immunological injury and the development of obliterative arteriopathy. Based on the findings, it is not unreasonable to raise the testable hypothesis that direct presentation of alloantigen by donor antigen-presenting cells is required for long-term, chronic-rejection-free allograft acceptance. In addition, chronic intermittent lymphatic disruption is implicated as a possible mechanism for the association between chronic interstitial allograft inflammation and the development of obliterative arteriopathy

Chronic liver allograft rejection in a population treated primarily with tacrolimus as baseline immunosuppression: Long-term follow-up and evaluation of features for histopathological staging

Background: Predisposing factors, long-term occurrence, and histopathological changes associated with recovery or progression to allograft failure from chronic rejection (CR) were studied in adult patients treated primarily with tacrolimus. Methods: CR cases were identified using stringent criteria applied to a retrospective review of computerized clinicopathological data and slides. Results: After 1973 days median follow- up, 35 (3.3%) of 1049 primary liver allograft recipients first developed CR between 16 and 2532 (median 242) days. The most significant risk factors for CR were the number (P40 years (P<0.03). Other demographic and matching parameters were not associated with CR in this cohort. Ten patients died with, but not of, CR. Eight required retransplantation because of CR at a median of 268 days. Ten resolved either histologically or by normalization of liver injury tests over a median of 548 days. CR persisted for 340 to 2116 days in the remaining seven patients. More extensive bile duct loss (P<0.01), small arterial loss (p<0.03), foam cell clusters (P<0.01) and higher total bilirubin (p<0.02) and aspartate aminotransferase (p<0.03) were associated with allograft failure from CR. Conclusions: Early chronic liver allograft rejection is potentially reversible and a combination of histological, clinical and laboratory data can be use to stage CR. Unique immunological and generative properties of liver allografts, which lead to low incidence and reversibility of early CR, can provide insights into transplantation biology

Isolation and primary cultures of human intrahepatic bile ductular epithelium

A technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments. © 1988 Tissue Culture Association, Inc

Exocomets size distribution in the β Pictoris planetary system

Stars and planetary system

Involvement of the Modifier Gene of a Human Mendelian Disorder in a Negative Selection Process

BACKGROUND:Identification of modifier genes and characterization of their effects represent major challenges in human genetics. SAA1 is one of the few modifiers identified in humans: this gene influences the risk of renal amyloidosis (RA) in patients with familial Mediterranean fever (FMF), a Mendelian autoinflammatory disorder associated with mutations in MEFV. Indeed, the SAA1 alpha homozygous genotype and the p.Met694Val homozygous genotype at the MEFV locus are two main risk factors for RA. METHODOLOGY/PRINCIPAL FINDINGS:HERE, WE INVESTIGATED ARMENIAN FMF PATIENTS AND CONTROLS FROM TWO NEIGHBORING COUNTRIES: Armenia, where RA is frequent (24%), and Karabakh, where RA is rare (2.5%). Sequencing of MEFV revealed similar frequencies of p.Met694Val homozygotes in the two groups of patients. However, a major deficit of SAA1 alpha homozygotes was found among Karabakhian patients (4%) as compared to Armenian patients (24%) (p = 5.10(-5)). Most importantly, we observed deviations from Hardy-Weinberg equilibrium (HWE) in the two groups of patients, and unexpectedly, in opposite directions, whereas, in the two control populations, genotype distributions at this locus were similar and complied with (HWE). CONCLUSIONS/SIGNIFICANCE:The excess of SAA1alpha homozygotes among Armenian patients could be explained by the recruitment of patients with severe phenotypes. In contrast, a population-based study revealed that the deficit of alpha/alpha among Karabakhian patients would result from a negative selection against carriers of this genotype. This study, which provides new insights into the role of SAA1 in the pathophysiology of FMF, represents the first example of deviations from HWE and selection involving the modifier gene of a Mendelian disorder

Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target