Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats

Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN), In vitro studies demonstrate the pivotal role of extracellular signal-regulated kinase (ERK) in the regulation of cellular proliferation in response to extracellular mitogens. In this study, we examined whether this kinase, as a convergence point of mitogenic stimuli, is activated in proliferative GN in vivo, Two different proliferative forms of anti-glomerular basal membrane (GEM) GN in rats were induced and whole cortical tissue as well as isolated glomeruli examined using kinase activity assays and Western blot analysis, Administration of rabbit anti-rat GEM serum to rats, preimmunized with rabbit IgG, induced an accelerated crescentic anti-GEM GN. A significant increase in cortical, and more dramatically glomerular ERK activity was detected at 1, 3, and 7 d after induction of GN, Immunization of Wistar-Kyoto rats with bovine GEM also induced a crescentic anti-GBM GN with an increase of renal cortical ERK activity after 4, 6, and 8 wk, ERK is phosphorylated and activated by the MAP kinase/ERK kinase (MEK), We detected a significant increase in the expression of glomerular MEK in the accelerated form of anti-GEM CN, providing a possible mechanism of long-term activation of ERK in this disease model, In contrast to ERK, activation of stress-activated protein kinase was only detectable at early stages of proliferative GN, indicating these related kinases to serve distinct roles in the pathogenesis of GN, Our observations point to ERK as a putative mediator of the proliferative response to immune injury in GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Donor lymphocyte infusions (DLI) have been shown to enhance the graft-versus-leukaemia (GVL) effect and induce haematological and molecular remission in patients with relapsed CML following allogeneic bone marrow transplantation (BMT). The potent donor cell-mediated cytolysis following DLI may lead to a short period of aplasia before the re-establishment of donor haematopoiesis. The absence of detectable donor cells in patients prior to DLI infusion may result in permanent aplasia in certain patients. We report on four patients who relapsed 1, 3, 6.5 and 7 years post-BMT for chronic phase CML and were treated with DLI from their original BMT donor. Polymorphic short tandem repeats (STRs) were used to assess haematological chimaerism both prior to and following DLI. At the time of relapse, STR-PCR indicated the presence of donor cells in all four patients, at levels ranging from 1-40%. A clinical and molecular response was seen in 4/4 patients following a short period of cytopenia and all patients remain in clinical remission with a follow-up of 2 months-3 years post-DLI. STR-PCR indicated that a response was occurring during the period of pancytopenia when metaphase analysis was unsuccessful. Lineage-specific analysis of the cellular response to DLI was monitored using STR-PCR of peripheral blood (PB) and bone marrow (BM) lymphocyte-enriched fractions and CD2-positive and -negative T cell fractions. In one patient BM and PB CD34-positive and -negative fractions were also assessed. A change in the ratio of donor:recipient cells in the PB lymphocyte fraction was the earliest molecular indication of an anti-leukaemic response. Subsequent conversion to donor chimaerism occurred in the other lineages and the granulocyte fraction was the last lineage to convert. In conclusion, lineage-specific STR-PCR permits detailed monitoring of subtle changes in donor/recipient cell dynamics in specific lineages following DLI during the crucial pancytopenic phase and may be a useful predictor of haematological response to DLI therapy.

FKBPL regulates estrogen receptor signaling and determines response to endocrine therapy

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor a (ERa) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERa phosphorylation on Ser118 in response to 17ß-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.

eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage

Purpose: In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina.

Methods: Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA.

Results: In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels.

Conclusions: In hyperoxia, reduced BH bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia. © 2012 The Association for Research in Vision and Ophthalmology, Inc.

Changes in lymphocyte subsets, interleukin 2, and soluble interleukin 2 receptor in old and very old age

In this study, the changes in some of the cellular components of the immune system and the activity of the cytokine interleukin 2, important for immune activation and lymphocyte proliferation, were measured in a large cross-sectional study of all age groups including octogenarian and nonagenarian subjects. In 206 apparently well community-living subjects, the absolute lymphocyte count and T and B cell numbers fell a little in old and very old subjects. Within the T cell compartment, helper/inducer CD4+ T cells, together with their subsets identified as 'naive' (CD4+/CD45RA+) and 'memory' (CD4+/CD45RO+) cells, also showed a decline with increased age. The suppressor/cytotoxic CD8+ subset showed no age-related change. The levels of the cytokine interleukin 2 were very low in octogenarian and nonagenarian subjects, while the soluble interleukin 2 receptor levels increased with increasing age. The interleukin 2 levels were associated with number and percentage of the 'memory' (CD4+/CD45RO+) subset of T cells which mediates the host response to previously met antigens. Since the interleukin 2 values were very low in the oldest groups and were associated with a reduced 'memory' (CD4+/CD45RO+) compartment, this suggests a possible mechanism of why the very elderly subject is more susceptible to morbidity and mortality from infectious or other agents.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 mu g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1 beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1. (C) 2008 Elsevier Inc. All rights reserved.

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.

Lymphocyte subsets in conjunctival mucosa-associated-lymphoid-tissue after exposure to retinal-S-antigen

Purpose: To evaluate the immune cell subsets in conjunctival mucosa-associated-lymphoid-tissue (C-MALT) following challenge with antigen. Methods: Ten adult female Lewis rats were studied. Five rats received one drop (5 µL) of retinal S-antigen (500 µg/mL in phosphate buffered saline, PBS) instilled into the lower fornix twice daily for 10 consecutive days. Five rats received PBS only and served as controls for the experiment. Two days after the last instillation the animals were sacrificed and the orbital contents prepared for immunohistological staining. A panel of monoclonal antibodies was used: CD5, CD4, CD8, CD25, and CD45RA. The number of positive cells were counted in sections of epibulbar, forniceal, and tarsal conjunctiva. Results: There was a significant increase in the number of CD8 T lymphocytes in the conjunctiva of animals receiving retinal S-antigen when compared to control animals. Conclusion: Conjunctival instillation of retinal S-antigen causes an immune response in the C-MALT with a significant increase in the CD8 T lymphocyte subset in this tissue. This response may be involved in the induction of tolerance to the encountered antigen.

Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress

In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair.

Effect of ultraviolet-B light on lymphocyte activity at doses at which normal bone marrow stem cells are preserved

Ultraviolet B (UVB) light is known to be immunosuppressive, but, probably because of a small UVC component in the emission spectra of some of the UVB lamps used, reports vary on effective dose levels. To prevent potentially lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, alloreactive donor T-cell activity must be suppressed. In this study, a narrow wavelength UVB lamp (TL01, 312 nm peak emission) was used to determine what doses of UVB were required to abolish rat lymphocyte proliferation while simultaneously preserving rat bone marrow progenitor cell and primitive hematopoietic stem cell viability. Lymphocyte proliferation, as measured by 3H-Thymidine incorporation, in response to lectin stimulation was abolished below detection at doses greater than 3,500 J/m2. When T-cell clonogenicity was measured in a limiting dilution assay, a small fraction (0.6%) was maintained at doses up to 4,000 J/m2. Cytotoxic T-lymphocyte (CTL) activity was reduced after treatment with 4,000 J/m2, but a significant level of cytotoxicity was still maintained. Natural killer cell cytolytic activity was not affected by doses up to 4,000 J/m2. At 4,000 J+m2 there was a 10% survival of colony-forming units-granulocyte-macrophage; a 1% and 4% survival of day-8 and day-12 colony-forming units-spleen, respectively; and 11% survival of marrow repopulating ability cells. Up to 25% of late cobblestone area forming cells (4 to 5 weeks), reflecting the more immature hematopoietic stem cells, were preserved in bone marrow treated with 4,000 J/m2, indicating that early stem cells are less sensitive to UVB damage than are more committed progenitor cells. Thus, a potential therapeutic window was established at approximately 4,000 J/m2 using this light source, whereby the potentially GVHD-inducing T cells were suppressed, but a sufficient proportion of the cells responsible for engraftment was maintained.