A flow cytometry based oligotrophic pollutant exposure test to detect bacterial growth inhibition and cell injury.

Toxicity of chemical pollutants in aquatic environments is often addressed by assays that inquire reproductive inhibition of test microorganisms, such as algae or bacteria. Those tests, however, assess growth of populations as a whole via macroscopic methods such as culture turbidity or colony-forming units. Here we use flow cytometry to interrogate the fate of individual cells in low-density populations of the bacterium Pseudomonas fluorescens SV3 exposed or not under oligotrophic conditions to a number of common pollutants, some of which derive from oil contamination. Cells were stained at regular time intervals during the exposure assay with fluorescent dyes that detect membrane injury (i.e., live-dead assay). Reduction of population growth rates was observed upon toxicant insult and depended on the type of toxicant. Modeling and cell staining indicate that population growth rate decrease is a combined effect of an increased number of injured cells that may or may not multiply, and live cells dividing at normal growth rates. The oligotrophic assay concept presented here could be a useful complement for existing biomarker assays in compliance with new regulations on chemical effect studies or, more specifically, for judging recovery after exposure to fluctuating toxicant conditions.

Caspase-induced inactivation of the anti-apoptotic TRAF1 during Fas ligand-mediated apoptosis.

The activation of the transcription factor NF-kappaB often results in protection against apoptosis. In particular, pro-apoptotic tumor necrosis factor (TNF) signals are blocked by proteins that are induced by NF-kappaB such as TNFR-associated factor 1 (TRAF1). Here we show that TRAF1 is cleaved after Asp-163 when cells are induced to undergo apoptosis by Fas ligand (FasL). The C-terminal cleavage product blocks the induction of NF-kappaB by TNF and therefore functions as a dominant negative (DN) form of TRAF1. Our results suggest that the generation of DN-TRAF1 is part of a pro-apoptotic amplification system to assure rapid cell death.

Danger signaling through the inflammasome acts as a master switch between tolerance and sensitization.

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.

Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus.

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.

IL-12 controls cytotoxicity of a novel subset of self-antigen-specific human CD28+ cytolytic T cells.

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.

Role of prostacyclin versus peroxisome proliferator-activated receptor beta receptors in prostacyclin sensing by lung fibroblasts.

Prostacyclin and its mimetics are used therapeutically for the treatment of pulmonary hypertension. These drugs act via cell surface prostacyclin receptors (IP receptors); however, some of them can also activate the nuclear receptor peroxisome proliferator-activated receptor beta (PPARbeta). We examined the possibility that PPARbeta is a therapeutic target for the treatment of pulmonary hypertension. Using the newly approved (for pulmonary hypertension) prostacyclin mimetic treprostinil sodium, reporter gene assays for PPARbeta activation and measurement of lung fibroblast proliferation were analyzed. Treprostinil sodium was found to activate PPARbeta in reporter gene assays and to inhibit proliferation of human lung fibroblasts at concentrations consistent with an effect on PPARs but not on IP receptors. The effects of treprostinil sodium on human lung cell proliferation are mimicked by those of the highly selective PPARbeta ligand GW0742. There are no receptor antagonists for PPARbeta or for IP receptors, but by using lung fibroblasts cultured from mice lacking PPARbeta (PPARbeta-/-) or IP (IP-/-), we demonstrate that the antiproliferative effects of treprostinil sodium are mediated by PPARbeta and not IP in lung fibroblasts. These observations suggest that some of the local, longer-term benefits of treprostinil sodium on reducing the remodeling associated with pulmonary hypertension may be mediated by PPARbeta. This study is the first to identify PPARbeta as a potential therapeutic target for the treatment of pulmonary hypertension, which is important because orally active PPARbeta ligands have been developed for the treatment of dyslipidemia.

Unusual astrocyte reactivity caused by the food mycotoxin ochratoxin A in aggregating rat brain cell cultures.

Ochratoxin A (OTA), a mycotoxin and widespread food contaminant, is known for its patent nephrotoxicity and potential neurotoxicity. Previous observations in vitro showed that in the CNS, glial cells were particularly sensitive to OTA. In the search for the molecular mechanisms underlying OTA neurotoxicity, we investigated the relationship between OTA toxicity and glial reactivity, in serum-free aggregating brain cell cultures. Using quantitative reverse transcriptase-polymerase chain reaction to analyze changes in gene expression, we found that in astrocytes, non cytotoxic concentrations of OTA down-regulated glial fibrillary acidic protein, while it up-regulated vimentin and the peroxisome proliferator-activated receptor-gamma expression. OTA also up-regulated the inducible nitric oxide synthase and the heme oxygenase-1. These OTA-induced alterations in gene expression were more pronounced in cultures at an advanced stage of maturation. The natural peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-delta(12,14) prostaglandin J2, and the cyclic AMP analog, bromo cyclic AMP, significantly attenuated the strong induction of peroxisome proliferator-activated receptor-gamma and inducible nitric oxide synthase, while they partially reversed the inhibitory effect of OTA on glial fibrillary acidic protein. The present results show that OTA affects the cytoskeletal integrity of astrocytes as well as the expression of genes pertaining to the brain inflammatory response system, and suggest that a relationship exists between the inflammatory events and the cytoskeletal changes induced by OTA. Furthermore, these results suggest that, by inducing an atypical glial reactivity, OTA may severely affect the neuroprotective capacity of glial cells.

Préservation de la fertilité masculine et cancer [Fertility preservation and cancer in the male].

Improvement in cancer treatments resulted in an increased number of men surviving cancer. Quality of life has become an important issue in these patients. Anti cancer treatments might have transient or definitive harmful effects on male fertility. Sperm cryoconservation is currently the only proven method to preserve fertility in patients undergoing oncologic treatment. It should be proposed to every patient at reproductive age before chemotherapy, radiotherapy or any surgery involving reproductive tract. Despite low use rate, this simple method could allow patients presenting infertility after treatment to father a child.

Neuroprotective role of lactate after cerebral ischemia.

It is well established that lactate can be used as an energy substrate by the brain by conversion to pyruvate and a subsequent oxidation in the mitochondria. Knowing the need for readily metabolizable substrates directly after ischemia and the protective effect of lactate after excitotoxicity, the aim of this study was to investigate whether lactate administration directly after ischemia could be neuroprotective. In vitro, the addition of 4 mmol/L L-lactate to the medium of rat organotypic hippocampal slices, directly after oxygen and glucose deprivation (OGD), protected against neuronal death, whereas a higher dose of 20 mmol/L was toxic. In vivo, after middle cerebral artery occlusion in the mouse, an intracerebroventricular injection of 2 microL of 100 mmol/L L-lactate, immediately after reperfusion, led to a significant decrease in lesion size, which was more pronounced in the striatum, and an improvement in neurologic outcome. A later injection 1 h after reperfusion did not reduce lesion size, but significantly improved neurologic outcome, which is an important point in the context of a potential clinical application. Therefore, a moderate increase in lactate after ischemia may be a therapeutic tool.

Peroxisome proliferator-activated receptor-beta as a target for wound healing drugs.

Healing of cutaneous wounds, which is crucial for survival after an injury, proceeds via a well-tuned pattern of events including inflammation, re-epithelialisation, and matrix and tissue remodelling. These events are regulated spatio-temporally by a variety of growth factors and cytokines. The inflammation that immediately follows injury increases the expression of peroxisome proliferator-activated receptor (PPAR)-beta in the wound edge keratinocytes and triggers the production of endogenous PPARbeta ligands that activate the newly produced receptor. This elevated PPARbeta activity results in increased resistance of the keratinocytes to the apoptotic signals released during wounding, allowing faster re-epithelialisation. The authors speculate that, in parallel, ligand activation of PPARbeta in infiltrated macrophages attenuates the inflammatory response, which also promotes repair. Thus, current understanding of the roles of PPARbeta in different cell types implicated in tissue repair has revealed an intriguing intercellular cross-talk that coordinates, spatially and temporally, inflammation, keratinocyte survival, proliferation and migration, which are all essential for efficient wound repair. These novel insights into the orchestrating roles of PPARbeta during wound healing may be helpful in the development of drugs for acute and chronic wound disorders.

GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

Effect of antenatal corticosteroids on fetal growth and gestational age at birth.

OBJECTIVE: To estimate the effect of multiple courses of antenatal corticosteroids on neonatal size, controlling for gestational age at birth and other confounders, and to determine whether there was a dose-response relationship between number of courses of antenatal corticosteroids and neonatal size. METHODS: This is a secondary analysis of the Multiple Courses of Antenatal Corticosteroids for Preterm Birth Study, a double-blind randomized controlled trial of single compared with multiple courses of antenatal corticosteroids in women at risk for preterm birth and in which fetuses administered multiple courses of antenatal corticosteroids weighed less, were shorter, and had smaller head circumferences at birth. All women (n=1,858) and children (n=2,304) enrolled in the Multiple Courses of Antenatal Corticosteroids for Preterm Birth Study were included in the current analysis. Multiple linear regression analyses were undertaken. RESULTS: Compared with placebo, neonates in the antenatal corticosteroids group were born earlier (estimated difference and confidence interval [CI]: -0.428 weeks, CI -0.10264 to -0.75336; P=.01). Controlling for gestational age at birth and confounding factors, multiple courses of antenatal corticosteroids were associated with a decrease in birth weight (-33.50 g, CI -66.27120 to -0.72880; P=.045), length (-0.339 cm, CI -0.6212 to -0.05676]; P=.019), and head circumference (-0.296 cm, -0.45672 to -0.13528; P<.001). For each additional course of antenatal corticosteroids, there was a trend toward an incremental decrease in birth weight, length, and head circumference. CONCLUSION: Fetuses exposed to multiple courses of antenatal corticosteroids were smaller at birth. The reduction in size was partially attributed to being born at an earlier gestational age but also was attributed to decreased fetal growth. Finally, a dose-response relationship exists between the number of corticosteroid courses and a decrease in fetal growth. The long-term effect of these findings is unknown. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00187382. LEVEL OF EVIDENCE: II.

Two alpha1-adrenergic receptor subtypes regulating the vasopressor response have differential roles in blood pressure regulation.

To study the functional role of individual alpha1-adrenergic (AR) subtypes in blood pressure (BP) regulation, we used mice lacking the alpha1B-AR and/or alpha1D-AR with the same genetic background and further studied their hemodynamic and vasoconstrictive responses. Both the alpha1D-AR knockout and alpha1B-/alpha1D-AR double knockout mice, but not the alpha1B-AR knockout mice, had significantly (p < 0.05) lower levels of basal systolic and mean arterial BP than wild-type mice in nonanesthetized condition, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. All mutants showed a significantly (p < 0.05) reduced catecholamine-induced pressor and vasoconstriction responses. It is noteworthy that the infusion of norepinephrine did not elicit any pressor response at all in alpha1B-/alpha1D-AR double knockout mice. In an attempt to further examine alpha1-AR subtype, which is involved in the genesis or maintenance of hypertension, BP after salt loading was monitored by tail-cuff readings and confirmed at the endpoint by direct intra-arterial recording. After salt loading, alpha1B-AR knockout mice developed a comparable level of hypertension to wild-type mice, whereas mice lacking alpha1D-AR had significantly (p < 0.05) attenuated BP and lower levels of circulating catecholamines. Our data indicated that alpha1B- and alpha1D-AR subtypes participate cooperatively in BP regulation; however, the deletion of the functional alpha1D-AR, not alpha1B-AR, leads to an antihypertensive effect. The study shows differential contributions of alpha1B- and alpha1D-ARs in BP regulation.