Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels

This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.     

Differential functions of tissue factor in the trans-activation of cellular signalling pathways

In this study we examined the ability of tissue factor (TF) alone, or in conjunction with factor VIIa, factor Xa and TFPI in activating a number of key signalling pathways associated with cellular growth, stress and differentiation responses in human endothelial cells. We used luciferase reporter systems to demonstrate the activation of p42/44 MAPK by the TF-FVIIa complex, mediated via the PAR1 receptor. TF alone was capable of interacting with the cell surface and was sufficient to activate the JNK-SAPK pathway and subsequently AP-1, but the level of activation was enhanced by the activity of FXa on PAR1 and 2. Furthermore, the phosphorylated form of the transmembrane-cytoplasmic domain of TF was directly responsible for activation of these pathways. CREB activation occurred in response to TF-FVIIa in a non-protease dependent manner but was lowered on addition of FXa. Finally, NFkappaB activation occurred in response to FVIIa or FXa, with the latter exhibiting higher levels of activation. In conclusion, we have shown that TF is capable of activating differing signalling pathways, via more than one mechanism. The differential influence of TF is modified depending on the presence of other coagulation factors and ultimately acts as a deciding factor in the determination of cellular fate.     

High radiosensitivity of germ cells in human male fetus

CONTEXT: Germ cells formed during human fetal life are essential for fertility of the adult, and several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. However, factors inducing a genotoxic stress may also be implicated. OBJECTIVES: We investigated the effect of gamma-irradiation on the functions of human fetal testis during the first trimester of gestation by using an organ culture system. Then we focused on the role of the p53 pathway in the observed effects. RESULTS: Germ cells were highly sensitive to irradiation even at doses as low as 0.1 and 0.2 Gy. Indeed, for these doses, one third of germ cells died by apoptosis. Other germ cells were blocked in their cycle, but no repair seemed to occur, and longer culture with the highest dose used showed that they were destined to die. Sertoli cells were less affected, although their proliferation and the level of anti-Müllerian hormone were reduced. Irradiation had no effect on testosterone secretion or on the expression of steroidogenic enzymes by Leydig cells. After irradiation, p53 phosphorylated on serine 15 was detected from 1-24 h in all cell types. This activation of p53 was accompanied by an increase in mRNA levels of proapoptotic factors Bax and Puma, whereas that of antiapoptotic Bcl-2 remained unchanged. P21, which is responsible for cell cycle arrest, was also up-regulated 6, 30, and 72 h after irradiation. Finally, when we added pifithrin-alpha, a specific inhibitor of p53 functions, a significant decrease in irradiation-induced apoptosis in both germ and Sertoli cells was observed, indicating the involvement of the p53 pathway in irradiation-induced apoptosis. CONCLUSIONS: This study demonstrated here for the first time the great sensitivity of human fetal germ cells to genotoxic stress caused by ionizing radiation.     

A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity

BACKGROUND: Despite the availability of efficacious drugs, the success of treating hypertension is limited by patients' inconsistent drug intake. Immunization against angiotensin II may offer a valuable alternative to conventional drugs for the treatment of hypertension, because vaccines induce relatively long-lasting effects and do not require daily dosing. Here we describe the preclinical development and the phase I clinical trial testing of a virus-like particle (VLP)-based antihypertensive vaccine. METHODS AND RESULTS: An angiotensin II-derived peptide was conjugated to the VLP Qbeta (AngQb). AngQb was highly immunogenic in mice and rats. To test for efficacy, spontaneously hypertensive rats (SHR) were immunized with 400 microg AngQb or VLP alone. Group mean systolic blood pressure (SBP) was reduced by up to 21 mmHg (159 +/- 2 versus 180 +/- 5 mmHg, P < 0.001), and total angiotensin II levels (antibody-bound and free) were increased ninefold (85 +/- 20 versus 9 +/- 1 pmol/l, P = 0.002) compared with VLP controls. SHR treated with the angiotensin-converting enzyme (ACE) inhibitor ramipril (1 mg/kg per day by mouth) reached an SBP of 155 +/- 2 mmHg. Twelve healthy volunteers of a placebo-controlled randomized phase I trial were injected once with 100 microg AngQb. Angiotensin II-specific antibodies were raised in all subjects (100% responder rate) and AngQb was well tolerated. CONCLUSIONS: AngQb reduces blood pressure in SHR to levels obtained with an ACE inhibitor, and is immunogenic and well tolerated in humans. Therefore, vaccination against angiotensin II has the potential to become a useful antihypertensive treatment providing long-lasting effects and improving patient compliance.     

Long-term safety, effectiveness and quality of a generic fixed-dose combination of nevirapine, stavudine and lamivudine

We assessed the long-term safety, effectiveness and quality of a fixed-dose combination of nevirapine, stavudine and lamivudine (triomune). HIV-1-infected adults initially enrolled in a one-year, open-label, single-arm, multicentre trial in Cameroon were followed for 2 years. Our results support the safety and effectiveness of the triomune combination for first-line treatment of HIV infection. Virological effectiveness appeared to wane somewhat during the second year of treatment, however, and plasma nevirapine concentrations were relatively high.     

Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance

Insulin and insulin-like growth factor 1 (IGF1) are ubiquitous growth factors that regulate proliferation in most mammalian tissues including pancreatic islets. To explore the specificity of insulin receptors in compensatory beta-cell growth, we examined two models of insulin resistance. In the first model, we used liver-specific insulin receptor knockout (LIRKO) mice, which exhibit hyperinsulinemia without developing diabetes due to a compensatory increase in beta-cell mass. LIRKO mice, also lacking functional insulin receptors in beta-cells (beta IRKO/LIRKO), exhibited severe glucose intolerance but failed to develop compensatory islet hyperplasia, together leading to early death. In the second model, we examined the relative significance of insulin versus IGF1 receptors in islet growth by feeding high-fat diets to beta IRKO and beta-cell-specific IGF1 receptor knockout (beta IGFRKO) mice. Although both groups on the high-fat diet developed insulin resistance, beta IRKO, but not beta IGFRKO, mice exhibited poor islet growth consistent with insulin-stimulated phosphorylation, nuclear exclusion of FoxO1, and reduced expression of Pdx-1. Together these data provide direct genetic evidence that insulin/FoxO1/Pdx-1 signaling is one pathway that is crucial for islet compensatory growth response to insulin resistance.     

A simple method for detection of major phenotypic abnormalities in myelodysplastic syndromes: expression of CD56 in CMML

Using a very simple flow cytometry protocol, we found that CD36 and CD117 on granulocytes and CD56 on monocytes were the major bone marrow phenotypic aberrations in patients with myelodysplasia, including CMML. CD56 on monocytes was associated with CMML. Importantly, phenotypic aberrations were lost on blood cells, except for CD56.     

Neural correlates of automatic beliefs about gender and race

Functional MRI was used to identify the brain areas underlying automatic beliefs about gender and race, and suppression of those attitudes. Participants (n = 20; 7 females) were scanned at 3 tesla while performing the Implicit Association Test (IAT), an indirect measure of race and gender bias. We hypothesized that ventromedial prefrontal cortex areas (PFC) would mediate gender and racial stereotypic attitudes, and suppression of these beliefs would recruit dorsolateral prefrontal cortex (DLPFC) and the anterior cingulate cortex (ACC). Performance data on the IAT revealed gender and racial biases. Racial bias was correlated with an explicit measure of racism. Results showed activation of anteromedial PFC and rostral ACC while participants implicitly made associations consistent with gender and racial biases. In contrast, associations incongruent with stereotypes recruited DLPFC. Implicit gender bias was correlated with amygdala activation during stereotypic conditions. Results suggest there are dissociable roles for anteromedial and dorsolateral PFC circuits in the activation and inhibition of stereotypic attitudes.     

Cotard's syndrome in adolescents and young adults is associated with an increased risk of bipolar disorder

OBJECTIVES: To assess the effect of age at onset on the phenomenology of Cotard's syndrome (CS) as a recent study reported a high rate of occurrence of bipolar disorder (BD) in adolescents and young adults with CS followed up for > or =2 years. METHODS: We reviewed all cases of CS reported since it was first described. A statistical analysis was carried out to determine the effect of age at onset on CS phenomenology. RESULTS: We found 138 cases including 21 cases aged 25 years or younger. In these younger CS patients, BD was more frequent, and the risk of associated BD was increased nine times (p < 0.0001). Within the BD sub-group (n = 27), admixture analysis identified two sub-groups with mean ages at onset of 18.7 years [standard deviation (SD) = 3.2] and 50.5 years (SD = 11.7). CONCLUSIONS: Young people with CS should be monitored carefully for the onset of BD, and families should be educated about this risk. Treatment with mood stabilizers can be helpful for those who develop BD. Within BD associated with CS, early versus late onset should be distinguished.