Predictive factors of adrenal insufficiency in patients admitted to acute medical wards: a case control study.

BACKGROUND: Adrenal insufficiency is a rare and potentially lethal disease if untreated. Several clinical signs and biological markers are associated with glucocorticoid failure but the importance of these factors for diagnosing adrenal insufficiency is not known. In this study, we aimed to assess the prevalence of and the factors associated with adrenal insufficiency among patients admitted to an acute internal medicine ward. METHODS: Retrospective, case-control study including all patients with high-dose (250 μg) ACTH-stimulation tests for suspected adrenal insufficiency performed between 2008 and 2010 in an acute internal medicine ward (n = 281). Cortisol values <550 nmol/l upon ACTH-stimulation test were considered diagnostic for adrenal insufficiency. Area under the ROC curve (AROC), sensitivity, specificity, negative and positive predictive values for adrenal insufficiency were assessed for thirteen symptoms, signs and biological variables. RESULTS: 32 patients (11.4%) presented adrenal insufficiency; the others served as controls. Among all clinical and biological parameters studied, history of glucocorticoid withdrawal was the only independent factor significantly associated with patients with adrenal insufficiency (Odds Ratio: 6.71, 95% CI: 3.08 -14.62). Using a logistic regression, a model with four significant and independent variable was obtained, regrouping history of glucocorticoid withdrawal (OR 7.38, 95% CI [3.18 ; 17.11], p-value <0.001), nausea (OR 3.37, 95% CI [1.03 ; 11.00], p-value 0.044), eosinophilia (OR 17.6, 95% CI [1.02; 302.3], p-value 0.048) and hyperkalemia (OR 2.41, 95% CI [0.87; 6.69], p-value 0.092). The AROC (95% CI) was 0.75 (0.70; 0.80) for this model, with 6.3 (0.8 - 20.8) for sensitivity and 99.2 (97.1 - 99.9) for specificity. CONCLUSIONS: 11.4% of patients with suspected adrenal insufficient admitted to acute medical ward actually do present with adrenal insufficiency, defined by an abnormal response to high-dose (250 μg) ACTH-stimulation test. A history of glucocorticoid withdrawal was the strongest factor predicting the potential adrenal failure. The combination of a history of glucocorticoid withdrawal, nausea, eosinophilia and hyperkaliemia might be of interest to suspect adrenal insufficiency.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

BACKGROUND: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. RESULTS: We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. CONCLUSIONS: In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

Total exposure and exposure rate effects for alcohol and smoking and risk of head and neck cancer: a pooled analysis of case-control studies.

Although cigarette smoking and alcohol consumption increase risk for head and neck cancers, there have been few attempts to model risks quantitatively and to formally evaluate cancer site-specific risks. The authors pooled data from 15 case-control studies and modeled the excess odds ratio (EOR) to assess risk by total exposure (pack-years and drink-years) and its modification by exposure rate (cigarettes/day and drinks/day). The smoking analysis included 1,761 laryngeal, 2,453 pharyngeal, and 1,990 oral cavity cancers, and the alcohol analysis included 2,551 laryngeal, 3,693 pharyngeal, and 3,116 oval cavity cancers, with over 8,000 controls. Above 15 cigarettes/day, the EOR/pack-year decreased with increasing cigarettes/day, suggesting that greater cigarettes/day for a shorter duration was less deleterious than fewer cigarettes/day for a longer duration. Estimates of EOR/pack-year were homogeneous across sites, while the effects of cigarettes/day varied, indicating that the greater laryngeal cancer risk derived from differential cigarettes/day effects and not pack-years. EOR/drink-year estimates increased through 10 drinks/day, suggesting that greater drinks/day for a shorter duration was more deleterious than fewer drinks/day for a longer duration. Above 10 drinks/day, data were limited. EOR/drink-year estimates varied by site, while drinks/day effects were homogeneous, indicating that the greater pharyngeal/oral cavity cancer risk with alcohol consumption derived from the differential effects of drink-years and not drinks/day.

Radioimmunotherapy of human colon carcinoma by 131I-labelled monoclonal anti-CEA antibodies in a nude mouse model.

A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.

A new alternative method for testing skin irritation using a human skin model: a pilot study.

Studies assessing skin irritation to chemicals have traditionally used laboratory animals; however, such methods are questionable regarding their relevance for humans. New in vitro methods have been validated, such as the reconstructed human epidermis (RHE) model (Episkin®, Epiderm®). The comparison (accuracy) with in vivo results such as the 4-h human patch test (HPT) is 76% at best (Epiderm®). There is a need to develop an in vitro method that better simulates the anatomo-pathological changes encountered in vivo. To develop an in vitro method to determine skin irritation using human viable skin through histopathology, and compare the results of 4 tested substances to the main in vitro methods and in vivo animal method (Draize test). Human skin removed during surgery was dermatomed and mounted on an in vitro flow-through diffusion cell system. Ten chemicals with known non-irritant (heptylbutyrate, hexylsalicylate, butylmethacrylate, isoproturon, bentazon, DEHP and methylisothiazolinone (MI)) and irritant properties (folpet, 1-bromohexane and methylchloroisothiazolinone (MCI/MI)), a negative control (sodiumchloride) and a positive control (sodiumlaurylsulphate) were applied. The skin was exposed at least for 4h. Histopathology was performed to investigate irritation signs (spongiosis, necrosis, vacuolization). We obtained 100% accuracy with the HPT model; 75% with the RHE models and 50% with the Draize test for 4 tested substances. The coefficients of variation (CV) between our three test batches were <0.1, showing good reproducibility. Furthermore, we reported objectively histopathological irritation signs (irritation scale): strong (folpet), significant (1-bromohexane), slight (MCI/MI at 750/250ppm) and none (isoproturon, bentazon, DEHP and MI). This new in vitro test method presented effective results for the tested chemicals. It should be further validated using a greater number of substances; and tested in different laboratories in order to suitably evaluate reproducibility.

Mesenteric fat-control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

Comparison of the polynomial model against explicit measurements of noise components for different mammography systems.

Given the adverse impact of image noise on the perception of important clinical details in digital mammography, routine quality control measurements should include an evaluation of noise. The European Guidelines, for example, employ a second-order polynomial fit of pixel variance as a function of detector air kerma (DAK) to decompose noise into quantum, electronic and fixed pattern (FP) components and assess the DAK range where quantum noise dominates. This work examines the robustness of the polynomial method against an explicit noise decomposition method. The two methods were applied to variance and noise power spectrum (NPS) data from six digital mammography units. Twenty homogeneously exposed images were acquired with PMMA blocks for target DAKs ranging from 6.25 to 1600 µGy. Both methods were explored for the effects of data weighting and squared fit coefficients during the curve fitting, the influence of the additional filter material (2 mm Al versus 40 mm PMMA) and noise de-trending. Finally, spatial stationarity of noise was assessed.Data weighting improved noise model fitting over large DAK ranges, especially at low detector exposures. The polynomial and explicit decompositions generally agreed for quantum and electronic noise but FP noise fraction was consistently underestimated by the polynomial method. Noise decomposition as a function of position in the image showed limited noise stationarity, especially for FP noise; thus the position of the region of interest (ROI) used for noise decomposition may influence fractional noise composition. The ROI area and position used in the Guidelines offer an acceptable estimation of noise components. While there are limitations to the polynomial model, when used with care and with appropriate data weighting, the method offers a simple and robust means of examining the detector noise components as a function of detector exposure.

Genetic manipulation of adult-born hippocampal neurons rescues memory in a mouse model of Alzheimer's disease.

In adult mammals, neural progenitors located in the dentate gyrus retain their ability to generate neurons and glia throughout lifetime. In rodents, increased production of new granule neurons is associated with improved memory capacities, while decreased hippocampal neurogenesis results in impaired memory performance in several memory tasks. In mouse models of Alzheimer's disease, neurogenesis is impaired and the granule neurons that are generated fail to integrate existing networks. Thus, enhancing neurogenesis should improve functional plasticity in the hippocampus and restore cognitive deficits in these mice. Here, we performed a screen of transcription factors that could potentially enhance adult hippocampal neurogenesis. We identified Neurod1 as a robust neuronal determinant with the capability to direct hippocampal progenitors towards an exclusive granule neuron fate. Importantly, Neurod1 also accelerated neuronal maturation and functional integration of new neurons during the period of their maturation when they contribute to memory processes. When tested in an APPxPS1 mouse model of Alzheimer's disease, directed expression of Neurod1 in cycling hippocampal progenitors conspicuously reduced dendritic spine density deficits on new hippocampal neurons, to the same level as that observed in healthy age-matched control animals. Remarkably, this population of highly connected new neurons was sufficient to restore spatial memory in these diseased mice. Collectively our findings demonstrate that endogenous neural stem cells of the diseased brain can be manipulated to become new neurons that could allow cognitive improvement.

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

A dynamic model for stem cell homeostasis and patterning in Arabidopsis meristems.

Plants maintain stem cells in their meristems as a source for new undifferentiated cells throughout their life. Meristems are small groups of cells that provide the microenvironment that allows stem cells to prosper. Homeostasis of a stem cell domain within a growing meristem is achieved by signalling between stem cells and surrounding cells. We have here simulated the origin and maintenance of a defined stem cell domain at the tip of Arabidopsis shoot meristems, based on the assumption that meristems are self-organizing systems. The model comprises two coupled feedback regulated genetic systems that control stem cell behaviour. Using a minimal set of spatial parameters, the mathematical model allows to predict the generation, shape and size of the stem cell domain, and the underlying organizing centre. We use the model to explore the parameter space that allows stem cell maintenance, and to simulate the consequences of mutations, gene misexpression and cell ablations.

Contribution of the gap junction proteins Connexin40 and Connexin43 to the control of blood pressure

Summary Cells in tissues and organs coordinate their activities by communicating with each other through intercellular channels named gap junctions. These channels are conduits between the cytoplasmic compartments of adjacent cells, allowing the exchange of small molecules which may be crucial for hormone secretion. Renin is normally secreted in a regulated manner by specific cells of the juxtaglomerular apparatus located within the renal cortex. Gap junctional communication may be requisite to maintain an accurate functioning in coordination of renin-producing cells, more especially as renin is of paramount importance for the control of blood pressure. Connexin43 (Cx43) and Cx40 form gap junctions that link in vivo the cells of the juxtaglomerular apparatus. Cx43 links the endothelial cells, whereas gap junctions made of Cx40 connect the endothelial cells, the renin secreting cells, as well as the endothelial cells of to the renin-secreting cells of the afferent arteriole. The observation that loss of Cx40 results in chronic hypertension associated with altered vasomotion and signal conduction along arterioles, has lead us to suggest that connexins may contribute to control blood pressure by participating to the integration of various mechanical, osmotic and electrochemical stimuli involved in the control of renin secretion and by mediating the adaptive changes of the vascular wall induced by elevated blood pressure and mechanical stress. We therefore postulated that the absence of Cx40 could have deleterious effects on the coordinated functioning of the renin-containing cells, hence accounting for hypertension. In the first part of my thesis, we reported that Cx40-deficient mice (Cx40) are hypertensive due to increased plasma renin levels and numbers of renin-producing cells. Besides, we demonstrated that prostaglandins and nitric oxide, which are possible mediators in the regulation of renin secretion by the macula densa, exert a critical role in the mechanisms controlling blood pressure ín Cx40 knockout hypertensive mice. In view of previous studies that stated avessel-specifc increase in the expression of Cx43 during renin-dependent hypertension, we hypothesized that Cx43 channels are particularly well-matched to integrate the response of cells constituting the vascular wall to hypertensive conditions. Using transgenic mice in which Cx43 was replaced by Cx32, we revealed that the replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin and prevent the renin-dependent hypertension which is normally induced in the 2K1C model. To gain insights into the regulation of connexins in two separate tissues exposed to the same fluid pressure, the second part of my thesis work was dedicated to the study of the impact of chronic hypertension and related hypertrophy on the expression of the cardiovascular connexins (Cx40, Cx37, Cx43 and Cx45) in mouse aorta and heart. Our results documented that the expression of connexins is differentially regulated in mouse aorta. according to the models of hypertension. Thus, blood pressure induces mechanical forces that differentially alter the expression of vascular connexins in order to respond to an adaptation of the aortic wall observed under pathological conditions. Altogether these data provide the first evidences that intercellular communication mediated by gap junctions is required for a proper renin secretion from the juxtaglomerular apparatus in order to control blood pressure.

Local Renal Circadian Clocks Control Fluid-Electrolyte Homeostasis and BP.

The circadian timing system is critically involved in the maintenance of fluid and electrolyte balance and BP control. However, the role of peripheral circadian clocks in these homeostatic mechanisms remains unknown. We addressed this question in a mouse model carrying a conditional allele of the circadian clock gene Bmal1 and expressing Cre recombinase under the endogenous Renin promoter (Bmal1(lox/lox)/Ren1(d)Cre mice). Analysis of Bmal1(lox/lox)/Ren1(d)Cre mice showed that the floxed Bmal1 allele was excised in the kidney. In the kidney, BMAL1 protein expression was absent in the renin-secreting granular cells of the juxtaglomerular apparatus and the collecting duct. A partial reduction of BMAL1 expression was observed in the medullary thick ascending limb. Functional analyses showed that Bmal1(lox/lox)/Ren1(d)Cre mice exhibited multiple abnormalities, including increased urine volume, changes in the circadian rhythm of urinary sodium excretion, increased GFR, and significantly reduced plasma aldosterone levels. These changes were accompanied by a reduction in BP. These results show that local renal circadian clocks control body fluid and BP homeostasis.

Genetic association tests: a method for the joint analysis of family and case-control data.

With the trend in molecular epidemiology towards both genome-wide association studies and complex modelling, the need for large sample sizes to detect small effects and to allow for the estimation of many parameters within a model continues to increase. Unfortunately, most methods of association analysis have been restricted to either a family-based or a case-control design, resulting in the lack of synthesis of data from multiple studies. Transmission disequilibrium-type methods for detecting linkage disequilibrium from family data were developed as an effective way of preventing the detection of association due to population stratification. Because these methods condition on parental genotype, however, they have precluded the joint analysis of family and case-control data, although methods for case-control data may not protect against population stratification and do not allow for familial correlations. We present here an extension of a family-based association analysis method for continuous traits that will simultaneously test for, and if necessary control for, population stratification. We further extend this method to analyse binary traits (and therefore family and case-control data together) and accurately to estimate genetic effects in the population, even when using an ascertained family sample. Finally, we present the power of this binary extension for both family-only and joint family and case-control data, and demonstrate the accuracy of the association parameter and variance components in an ascertained family sample.

Systemic in vivo lentiviral delivery of miR-15a/16 reduces malignancy in the NZB de novo mouse model of chronic lymphocytic leukemia.

Similar to human chronic lymphocytic leukemia (CLL), the de novo New Zealand Black (NZB) mouse model has a genetically determined age-associated increase in malignant B-1 clones and decreased expression of microRNAs miR-15a and miR-16 in B-1 cells. In the present study, lentiviral vectors were employed in vivo to restore miR-15a/16, and both the short-term single injection and long-term multiple injection effects of this delivery were observed in NZB. Control lentivirus without the mir-15a/16 sequence was used for comparison. We found that in vivo lentiviral delivery of mir-15a/16 increased miR-15a/16 expression in cells that were transduced (detected by GFP expression) and in sera when compared with control lentivirus treatment. More importantly, mice treated with the miR-expressing lentivirus had decreased disease. The lentivirus had little systemic toxicity while preferentially targeting B-1 cells. Short-term effects on B-1 cells were direct effects, and only malignant B-1 cells transduced with miR-15a/16 lentivirus had decreased viability. In contrast, long-term studies suggested both direct and indirect effects resulting from miR-15a/16 lentivirus treatment. A decrease in B-1 cells was found in both the transduced and non-transduced populations. Our data support the potential use of systemic lentiviral delivery of miR-15a/16 to ameliorate disease manifestations of CLL.

Current status of a model system: the gene Gp-9 and its association with social organization in fire ants.

The Gp-9 gene in fire ants represents an important model system for studying the evolution of social organization in insects as well as a rich source of information relevant to other major evolutionary topics. An important feature of this system is that polymorphism in social organization is completely associated with allelic variation at Gp-9, such that single-queen colonies (monogyne form) include only inhabitants bearing B-like alleles while multiple-queen colonies (polygyne form) additionally include inhabitants bearing b-like alleles. A recent study of this system by Leal and Ishida (2008) made two major claims, the validity and significance of which we examine here. After reviewing existing literature, analyzing the methods and results of Leal and Ishida (2008), and generating new data from one of their study sites, we conclude that their claim that polygyny can occur in Solenopsis invicta in the U.S.A. in the absence of expression of the b-like allele Gp-9(b) is unfounded. Moreover, we argue that available information on insect OBPs (the family of proteins to which GP-9 belongs), on the evolutionary/population genetics of Gp-9, and on pheromonal/behavioral control of fire ant colony queen number fails to support their view that GP-9 plays no role in the chemosensory-mediated communication that underpins regulation of social organization. Our analyses lead us to conclude that there are no new reasons to question the existing consensus view of the Gp-9 system outlined in Gotzek and Ross (2007).