Increased rates of CD4+ and CD8+ T lymphocyte turnover in simian immunodeficiency virus-infected macaques

Defining the rate at which T cells turn over has important implications for our understanding of T lymphocyte homeostasis and AIDS pathogenesis, yet little information on T cell turnover is available. We used the nucleoside analogue bromodeoxyuridine (BrdUrd) in combination with five-color flow cytometric analysis to evaluate T lymphocyte turnover rates in normal and simian immunodeficiency virus (SIV)-infected rhesus macaques. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labeling and elimination rates of both CD4+ and CD8+ BrdUrd-labeled cells were increased by 2- to 3-fold as compared with normal controls. In normal and SIV-infected animals, the rates of CD4+ T cell BrdUrd-labeling and decay were closely correlated with those of CD8+ T cells. The elimination rate of BrdUrd-labeled cells was accelerated in both naive and memory T lymphocytes in SIV-infected animals. Our results provide direct evidence for increased rates of both CD4+ and CD8+ T cell turnover in AIDS virus infection and have important implications for our understanding of T cell homeostasis and the mechanisms responsible for CD4+ T cell depletion in AIDS.

Retinoid-related orphan receptor γ (RORγ) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis

To identify the physiological functions of the retinoid-related orphan receptor γ (RORγ), a member of the nuclear receptor superfamily, mice deficient in RORγ function were generated by targeted disruption. RORγ−/− mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that RORγ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of RORγ−/− mice contains 74.4% ± 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of RORγ−/− mice. The latter was supported by the observed increase in annexin V-positive cells. RORγ−/− thymocytes placed in culture exhibit a dramatic increase in the rate of “spontaneous” apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORγ−/− mice. Our observations indicate that RORγ is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which RORγ negatively controls apoptosis in thymocytes.

Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy

Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP–/– fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP–/– fibroblasts were exposed for 3 weeks to 20 µM GPI 6150 (1,11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (Ki = 60 nM) and one of the most potent PARP inhibitors to date (IC50 = 0.15 µM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by ∼91%, to about the residual levels in PARP–/– cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.

Oxygen deprivation causes suspended animation in the zebrafish embryo

Continuous exposure to oxygen is essential for nearly all vertebrates. We found that embryos of the zebrafish Danio rerio can survive for 24 h in the absence of oxygen (anoxia, 0% O2). In anoxia, zebrafish entered a state of suspended animation where all microscopically observable movement ceased, including cell division, developmental progression, and motility. Animals that had developed a heartbeat before anoxic exposure showed no evidence of a heartbeat until return to terrestrial atmosphere (normoxia, 20.8% O2). In analyzing cell-cycle changes of rapidly dividing blastomeres exposed to anoxia, we found that no cells arrested in mitosis. This is in sharp contrast to similarly staged normoxic embryos that consistently contain more than 15% of cells in mitosis. Flow cytometry analysis revealed that blastomeres arrested during the S and G2 phases of the cell cycle. This work indicates that survival of oxygen deprivation in vertebrates involves the reduction of diverse processes, such as cardiac function and cell-cycle progression, thus allowing energy supply to be matched by energy demands.

Leukemia inhibitory factor induces differentiation of pituitary corticotroph function: an immuno-neuroendocrine phenotypic switch.

Leukemia inhibitory factor (LIF) promotes differentiated cell function in several systems. We recently reported LIF and LIF receptor expression in human fetal pituitary corticotrophs in vivo and demonstrated LIF stimulation of adrenocorticotrophin (ACTH) transcription in vitro, suggesting a role for LIF in corticotroph development. We therefore assessed the action of LIF on proliferating murine corticotroph cells (AtT20). LIF impairs proliferation of AtT20 cells (25% reduction versus control, P < 0.03), while simultaneously enhancing ACTH secretion (2-fold, P < 0.001) and augmenting ACTH responsiveness to corticotrophin-releasing hormone (CRH) action (4-fold, P < 0.001). This attenuation of cell growth is due to a block of cell cycle progression from G1 into S phase, as measured by flow cytometric analysis (24 +/- 0.8 versus 11.57 +/- 1.5, P < 0.001). Using bromodeoxyuridine incorporation assays, loss of cells in S phase was confirmed (25 +/- 0.08 to 9.4 +/- 1.4, P < 0.008). In contrast, CRH induced the G2/M phase (3.6 +/- 0.2 to 15.4 +/- 3, P < 0.001). This effect was blunted by LIF (P < 0.001 versus CRH alone). Cyclin A mRNA levels, which decline in S phase, were stimulated 3.5-fold by LIF and markedly suppressed by CRH. These results indicate a LIF-induced cell cycle block occurring at G1/S in corticotroph cells. Thus, LIF reduces proliferation, enhances ACTH secretion, and potentiates effects of CRH on ACTH secretion while blocking effects of CRH on the cell cycle. Responses of these three markers of differentiated corticotroph function indicate LIF to be a differentiation factor for pituitary corticotroph cells by preferential phenotypic switching from proliferative to synthetic.

Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia.

Lack of SHPTP1 results in src-family kinase hyperactivation and thymocyte hyperresponsiveness.

Protein tyrosine phosphorylation and dephosphorylation are key regulatory events in T-cell receptor (TCR) signaling. We investigated the role of the tyrosine phosphatase SHPTP1 in TCR signaling by analysis of TCR signal transduction in motheaten (me/me) mice, which lack SHPTP1 expression. As revealed by flow cytometric analysis, thymocyte development was normal in me/me mice. However, me/me thymocytes hyperproliferated (3-to 5-fold) in response to TCR stimulation, whereas their response to interleukin 2 stimulation was unchanged compared with normal thymocytes. TCR-induced hyperproliferation of me/me thymocytes was reproduced in purified single-positive thymocytes. Moreover, me/me thymocytes produced increased amounts of interleukin 2 production upon TCR stimulation. Biochemical analysis revealed that, in response to TCR or TCR/CD4 stimulation, thymocytes lacking SHPTP1 showed increased tyrosyl phosphorylation of several cellular substrates, which correlated with increased activation of the src-family kinases Lck and Fyn. Taken together, our data suggest that SHPTP1 is an important negative regulator of TCR signaling, acting at least in part to inactivate Lck and Fyn.

Prolactin increases CD4/CD8 cell ratio in thymus-grafted congenitally athymic nude mice.

One distinctive effect on T-cell development was analyzed by selectively increasing serum prolactin (PRL) concentration in thymus-grafted congenitally athymic nude mice and by neutralizing PRL in suspension cultures of thymus from 1-day-old neonatal mice. Flow cytometric analysis of single-positive CD4+ and CD8+ cells derived from inguinal lymph nodes revealed a CD4/CD8 cell ratio of 2.2 +/- 0.18 (mean +/- SEM) in thymus-grafted nude mice that is similar to the ratio for immune-competent BALB/c mice (2.0 +/- 0.06). Addition of the pituitary to thymus-grafted nude mice significantly elevated serum PRL (P < 0.005) and increased the CD4/CD8 cell ratio (2.8 +/- 0.12; P < 0.005), demonstrating preferential stimulation of CD4+ cell development. T cells in nude mice receiving sham (submandibular salivary gland) or pituitary grafts alone were below detectable levels. Suspension cultures of neonatal thymus treated with anti-mouse PRL antiserum resulted in 20% and 30% decreases in double-positive CD4+8+ thymocytes and thymocyte viability, respectively. A 10-fold increase in double-negative CD4-8- thymocytes expressing the interleukin 2 receptor alpha chain, CD25, was also observed concurrently. Our findings illustrate an important way in which PRL may participate in two interrelated mechanisms: the regulation of peripheral single-positive cells and the maintenance of thymocyte viability during the double-positive stage of intrathymic differentiation.

CD38 ligation induces tyrosine phosphorylation of Bruton tyrosine kinase and enhanced expression of interleukin 5-receptor alpha chain: synergistic effects with interleukin 5.

Mouse CD38 has been implicated in the regulation of both B-cell proliferation and protection of B cells from irradiation-induced apoptosis. CD38 ligation on B cells by CS/2, an anti-mouse CD38 monoclonal antibody, induced proliferation, IgM secretion, and tyrosine phosphorylation of Bruton tyrosine kinase in B cells from wild-type mice. B cells from X chromosome-linked immunodeficient mice did not respond at all to anti-CD38 antibody, although CD38 expression on these B cells was comparable to that on wild-type B cells. We infer from these results that Bruton tyrosine kinase activation is involved in B-cell triggering after cross-linkage of CD38. Analysis of the synergistic effects of various cytokines with CD38 ligation on B-cell activation revealed that interleukin 5 (IL-5) showed the most potent effect on B-cell proliferation, Blimp1 gene expression, and IgM production. These synergistic effects were not seen with B cells from X chromosome-linked immunodeficient mice. Flow cytometry analysis revealed that CD38 ligation increased surface expression of the IL-5-receptor alpha chain on B cells. These data indicate that CD38 ligation increases IL-5 receptor alpha expression and synergizes with IL-5 to enhance Blimp1 expression and IgM synthesis.

Detection of membrane-bound HLA-G translated products with a specific monoclonal antibody.

A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.

Caracterização e aplicação analítica de eletrodos modificados com sistemas porfirínicos supramoleculares

Estudos com eletrodos modificados foram conduzidos utilizando dois sistemas porfirínicos supramoleculares diferentes. O primeiro foi baseado na modificação de eletrodo de carbono vítreo com uma porfirina de níquel tetrarrutenada, [NiIITPyP{RuII(bipy)2Cl}4]4+. A modificação do eletrodo foi realizada por meio de sucessivos ciclos voltamétricos em meio alcalino (pH 13), gerando um eletrodo com característica similar a eletrodos modificados com α-Ni(OH)2. A caracterização química do filme formado foi realizada através das técnicas de voltametria cíclica, ressonância paramagnética eletrônica, espectroscopia eletrônica por reflectância e espectroscopia Raman com ensaio espectro-eletroquímico. Os resultados sugerem a formação de um polímero de coordenação, [µ-O2-NiIITPyP{RuII(bipy)2Cl}4]n, composto por subunidades porfirínicas ligadas entre si por pontes µ-peroxo axialmente coordenadas aos átomos de níquel (Ni-O-O-Ni). O crescimento do filme apresentou dependência da alcalinidade do meio pela formação do precursor octaédrico [Ni(OH)2TRPyP]2+ em solução, pela coordenação de OH- nas posições axiais do átomo de níquel. O processo de eletropolimerização indicou a participação de radical hidroxil, gerado por oxidação eletrocatalítica da água nos sítios periféricos da porfirina contendo o complexo de rutênio. O mesmo eletrodo foi aplicado como sensor eletroquímico para análise amperométrica de ácido fólico em comprimidos farmacêuticos. O sensor foi associado a um sistema de Batch Injection Analysis (BIA) alcançando considerável rapidez e baixo limite de detecção. Para as análises das amostras também foi proposto um método para a remoção da lactose, que agia como interferente. O segundo estudo envolveu a modificação de eletrodos de carbono vítreo com diferentes hemoglobinas, naturais (HbA0, HbA2 e HbS) e sintéticas (Hb-PEG5K2, αα-Hb-PEG5K2 e BT-PEG5K4), para a avaliação da eficiência na redução eletrocatalítica de nitrito mediada por FeI-heme. Os filmes foram produzidos pela mistura de soluções das hemoglobinas com brometo de didodecildimetiltrimetilamônio (DDAB), aplicados nas superfícies com consecutiva evaporação, formando filmes estáveis. Os valores de potencial redox para os processos do grupo heme e a sua associação com a disponibilidade do grupo na proteína foram avaliados por voltametria cíclica. Os valores das constantes de velocidade, k, para redução de nitrito foram obtidos por cronoamperometria em -1,1 V (vs Ag/AgCl(KCl 3M)) que foram utilizados para estudo comparativo entre as espécies sintéticas para eventual aplicação clínica.

Degradação de atrazina por processo foto-Fenton monitorado por injeção seqüencial e cromatografia a líquido de alta eficiência

Processos de fotodegradação de compostos orgânicos tóxicos têm sido bastante estudados. Este trabalho trata da aplicação do processo foto-Fenton para a degradação de atrazina em água (composto modelo). O efeito das concentrações dos seguintes compostos foi avaliado: peróxido de hidrogênio (2 a 6 mmol L-1) e ferrioxalato de potássio (0,2 a 1 mmol L-1). Os experimentos foram realizados em um reator com lâmpada UV - 8W (254nm). O processo de fotodegradação foi monitorado por medidas de espectrofotometria de absorção molecular automatizada por injeção seqüencial (SIA) para determinação de peróxido de hidrogênio e por cromatografia a líquido de alta eficiência (CLAE) para determinação de atrazina e metabólitos. Os experimentos demonstram que o processo de foto-Fenton é viável para o tratamento de atrazina em água.

Retinal microglia are activated by systemic fungal infection

Purpose: To determine whether systemic fungal infection could cause activation of retinal microglia and therefore could be potentially harmful for patients with retinal degenerative diseases. Methods: Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by (i) confocal immunofluorescence and (ii) flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII and anti-CD45 antibodies. Results: Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial re-location in retinal layers. Conclusions: As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration or retinitis pigmentosa.

In vitro differentiation of murine hematopoietic progenitor cells toward the myeloid lineage occurs in response to Staphylococcus aureus and yeast species

We have studied the effect of inactivated microbial stimuli (Candida albicans, Candida glabrata, Saccharomyces boulardii, and Staphylococcus aureus) on the in vitro differentiation of lineage negative (Lin−) hematopoietic progenitor mouse cells. Purified Lin− progenitors were co-cultured for 7 days with the stimuli, and cell differentiation was determined by flow cytometry analysis. All the stimuli assayed caused differentiation toward the myeloid lineage. S. boulardii and particularly C. glabrata were the stimuli that induced in a minor extent differentiation of Lin− cells, as the major population of differentiated cells corresponded to monocytes, whereas C. albicans and S. aureus induced differentiation beyond monocytes: to monocyte-derived dendritic cells and macrophages, respectively. Interestingly, signaling through TLR2 by its pure ligand Pam3CSK4 directed differentiation of Lin− cells almost exclusively to macrophages. These data support the notion that hematopoiesis can be modulated in response to microbial stimuli in a pathogen-dependent manner, being determined by the pathogen-associated molecular patterns and the pattern-recognition receptors involved, in order to generate the populations of mature cells required to deal with the pathogen.

Immune biomarkers in acute graft-versus-host disease

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014