Standardization and Cross Validation of Alloreactive IFNγ ELISPOT Assays Within the Clinical Trials in Organ Transplantation Consortium

Emerging evidence indicates memory donor‐reactive T cells are detrimental to transplant outcome and that quantifying the frequency of IFNγ‐producing, donor‐reactive PBMCs by ELISPOT has potential utility as an immune monitoring tool. Nonetheless, differences in assay performance among laboratories limit the ability to compare results. In an effort to standardize assays, we prepared a panel of common cellular reagent standards, developed and cross validated a standard operating procedure (SOP) for alloreactive IFNγ ELISPOT assays in several research laboratories supported by the NIH‐funded Clinical Trials in Organ Transplantation (CTOT) Consortium. We demonstrate that strict adherence to the SOP and centralized data analysis results in high reproducibility with a coefficient of variance (CV) of ～30%. This standardization of IFNγ ELISPOTassay will facilitate interpretation of data from multicenter transplantation research studies and provide the foundation for developing clinical laboratory testing strategies to guide therapeutic decision‐making in transplant patients.     

Parkinson's disease cybrids,differentiated or undifferentiated,maintain morphological and biochemical phenotypes different from those of control cybrids

SH‐SY5Y, control, and Parkinson's disease (PD) cybrids prepared from an Indian population were differentiated using retinoic acid (RA) for understanding their dopaminergic characteristics and neuritogenesis. Undifferentiated control and PD cybrids exhibited higher levels of TH mRNA, but lower c‐RET expression, short neurites, low neuritic density, and low proportion of cells with neurites compared with the undifferentiated parent cell line, SH‐SY5Y. The expression levels of DAT and Ptx3 were similar to SH‐SY5Y. PD cybrids showed poor viability and lower differentiating potency than SH‐SY5Y or control cybrids. RA treatment for 6 days elevated c‐RET expression and corrected the neuritic morphology of the control, but not of PD cybrids. Cell viability was found to be reduced in differentiated control and PD cybrids. TH expression level was significantly elevated in SH‐SY5Y following RA treatment, but not in both the cybrids. In differentiated control and PD cybrids, the TH immunofluorescence intensity was significantly lower compared with SH‐SY5Y cells. MitoTracker Green fluorescence intensity of the mitochondria was higher in differentiated PD cybrids. Dopamine released into the medium was unaffected in the differentiated SH‐SY5Y or in the control cybrids but was significantly elevated in PD cybrids. These results suggest that PD cybrids, differentiated or undifferentiated, maintained morphological and biochemical phenotypes significantly different from those of the control cybrids, or the differentiated SH‐SY5Y cells, and therefore could be an ideal cellular model of the disease for pharmacological screening of drugs and for investigation of the pathophysiology of PD. © 2013 Wiley Periodicals, Inc.     

Combined treatment with CD40 costimulation blockade, T-cell depletion, low-dose irradiation, and donor bone marrow transfusion in limb allograft survival

To determine the efficacy of a regimen based on CD40 costimulation blockade and donor bone marrow in the limb allograft model, C57Bl/6 mice received limb allografts from Balb/c mice and either no treatment or a combination of MR1 (anti-CD40 ligand monoclonal antibody), CD4+ and CD8+ T-cell-depleting antibodies, low-dose irradiation, and bone marrow transfusion from Balb/c donors for 1 or 2 weeks. Recipients treated for 1 week showed rejection at 38.2 +/- 5.4 (mean +/- SEM) days, while those treated for 2 weeks had allograft survival of 56.5 +/- 9.9, with a range up to 91 days. Histology demonstrated rejection which was less cell-mediated and suggestive of transplant vasculopathy. Differential rejection of skin occurred first. Thus, a combined regimen based on CD40 costimulatory blockade and donor marrow significantly prolonged allograft survival. However, tolerance was not achieved, and histology suggests chronic rejection as a possible cause of allograft loss.     

Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse‐1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2‐photon microscopy, we analyzed the effects of DNAse‐1 on NETs in mouse orthotopic lung allografts damaged by ischemia‐reperfusion injury. Although DNAse‐1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4⁺ T cells and donor‐derived antigen presenting cells. DNAse‐1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen‐specific CD4⁺ T cell proliferation through activating toll‐like receptor (TLR) — Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil‐specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse‐1 administration to wild‐type recipients promotes the recognition of allo‐ and self‐antigens and prevents immunosuppression‐mediated lung allograft acceptance through a MyD88‐dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.     

Melatonin inhibits 6-hydroxydopamine production in the brain to protect against experimental parkinsonism in rodents

Abstract:  We tested the hypothesis that melatonin regulates formation of 6-hydroxydopamine (6-OHDA) in the brain and thereby protects animals from dopaminergic neurotoxicity and the development of parkinsonism in animals. Employing a ferrous-ascorbate-dopamine (FAD) hydroxyl radical (^•OH) generating system, in the present study we demonstrate a dose-dependent attenuation of 6-OHDA generation by melatonin in vitro. Intra-median forebrain bundle infusion of FAD caused significant depletion of striatal dopamine (DA), which was blocked by melatonin. Per-oral administration of l -3,4-dihydroxyphenylalanine (l -DOPA) for 7 days caused a dose-dependent increase in the formation of 6-OHDA in the mouse striatum, which was increased synergistically by the systemic administration of the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the 7th day of l -DOPA treatment. Melatonin treatment significantly attenuated both the l -DOPA and MPTP-induced increases in the levels of striatal 6-OHDA, and protected against striatal DA depletion caused by the neurotoxin. These observations suggest a novel mode of melatonin-induced dopaminergic neuroprotection in two models of Parkinson's disease, and suggest the possible therapeutic use of this well-known antioxidant indoleamine neurohormone in parkinsonism.     

Effect of age on thrombosis and morbidity in the mouse-limb transplantation model

Murine models of limb transplantation have been recently described. Because of the technical challenge, non-technical factors that influence the success rate and are easily controlled should be considered. This study investigated the effect of recipient animal age on anastomotic thrombosis, morbidity, and mortality. Twelve allogeneic heterotopic hindlimb transplants were performed using the femoral vessels with end-to-end arterial and end-to-side venous anastomoses. Group 1 (n=8) consisted of 2 to 3-month-old mice weighing 17 to 20 g, and Group 2 (n=7) included 7 to 8-month-old mice weighing 24 to 27 g. In Group 1, 6/8 (75 percent) transplants were successful, while in Group 2, only 1/7 (14 percent) allografts survived (p<0.05). A statistically significant difference in the incidence of vascular compromise of the ipsilateral recipient hindlimb was also noted (p<0.01). The authors conclude that although smaller, juvenile, inbred mice have a higher anastomotic patency rate, with greater collateral vasculature in the hindlimbs, and are therefore more suitable for limb transplantation research.     

Inhibitory action of morphine on adenosine triphosphatase content in the whole and individual nuclei of mouse brain during the tolerance-dependence development and its reversal by naloxone

Fluctuations in the content and distribution of adenosine triphosphatase in the brain of mice during the period of morphine tolerance-dependence development as well as normal and and naloxone induced withdrawal have been studied. The histochemical investigation revealed the enzyme activity in the neurons, neuroglia and blood vessels. In the control animals the nucleus caudatus putamen, globus pallidus, hippocampus, nuclei of amygdala, nuclei hypothalami, substantia grisea centralis, griseum pontis, nucleus trapezoideus, nucleus prepositus hypoglossi, nucleus parabrachialis, nucleus vestibularis, nucleus nervi hypoglossi, nucleus dorsalis nervi vagi, nucleus olivaris and nucleus centralis superior are found to be very rich in ATPase. However, morphine treatment inhibited the enzyme in all the above nuclei and it was linear with the increase in dose and the duration of the treatment. Cytophotometric studies reveal that the differences in the enzymatic activity varies from nuclei to nuclei. Surprisingly enough, normal withdrawal as well as naloxone induced withdrawal significantly elevated the enzyme levels. All above findings have been confirmed biochemically. The study gives a firm support of the earlier finding that morphine inhibits ATPase. In addition to this, the present work also reveals a direct antagonistic effect of naloxone on the content of the enzyme in the morphine treated animals. This suggests that the observed inhibition of the enzyme is narcotic specific. The role of ATPase in the Na+, K+ transport is discussed with respect to morphine action. In the light of the present investigation, the effect of morphine on the neurotransmitter release, and the cause and effect there upon has been analyzed. It has been suggested that ATPase might be the enzyme responsible for the observed pharmacological responses of the neurons to the application of the drug by affecting the Na+, K+ flux and neurotransmitter release.     

Childhood lymphoblastic cancer: subgroups defined by nonhuman primate antisera to human lymphatic leukemia cell antigens.

Lymphoblasts from 23 children with acute lymphocytic leukemia (ALL) and 10 with lymphoblastic lymphoma (LBL) were studied by complement-dependent microcytoxicity tests with two nonhuman primate antisera defining leukemia-associated and lymphoma-associated antigens. Cells form 15 patients with ALL and 1 with LBL reacted only with antiserum to chronic lymphatic leukemia (CLL). These group-I patients were predominantly female. Most were pancytopenic and lacked mediastinal widening and T-cell markers; lymphoblasts from 15 were periodic acid-Schiff-positive. Cells from 8 male patients reacted only with antiserum to converted lymphosarcoma (LS). All these group-II patients expressed T-cell markers; 5 had mediastinal enlargement and 2, an abdominal mass. Six of the 8 were PAS-negative. Cells from 9 patients reacted with both antisera. The group-III patients demonstrated some characteristics of each of the above groups. Patients whose lymphoblasts reacted with CLL antiserum presented with clinical and laboratory features indicative of a good prognosis, i.e., ALL with PAS positivity and no T-cell markers or localized mass. Patients whose cells reacted with LS antiserum often had bad prognostic features: mediastinal or abdominal mass, expression of T-cell markers, and PAS negativity. These antisera appear able to differentiate childhood ALL from LBL. The distinction is important prognostically and perhaps therapeutically.     

Childhood leukemia with simultaneously expressed myeloid and lymphoid markers suggesting stem cell origin

A case study is presented of a leukemic patient whose cells express markers of both myeloid and lymphoid cells. Cells were identified from bone marrow which expressed either myeloid antigens, lymphoid antigens, or both myeloid and lymphoid antigens, indicating a possible common stem cell capable of differentiating along either a lymphoid or myeloid cell lineage. Using specific monoclonal antibodies, 40-70% of the cells were reactive with anti-T-cell antibodies, 50% of the cells were reactive with antibodies to the common ALL antigen (CALLA), and 80-90% of the cells were reactive with antibodies directed against myeloid antigens. Using double staining techniques, some cells were found to demonstrate only myeloid markers; others, only lymphoid markers; and others, both myeloid and lymphoid markers. These results suggest that a common stem cell is capable of differentiating along both lymphoid and myeloid lineages.