Androgen-dependent pathology demonstrates myopathic contribution to the Kennedy disease phenotype in a mouse knock-in model

Kennedy disease, a degenerative disorder characterized by androgen-dependent neuromuscular weakness, is caused by a CAG/glutamine tract expansion in the androgen receptor (Ar) gene. We developed a mouse model of Kennedy disease, using gene targeting to convert mouse androgen receptor (AR) to human sequence while introducing 113 glutamines. AR113Q mice developed hormone and glutamine length-dependent neuromuscular weakness characterized by the early occurrence of myopathic and neurogenic skeletal muscle pathology and by the late development of neuronal intranuclear inclusions in spinal neurons. AR113Q males unexpectedly died at 2-4 months. We show that this androgen-dependent death reflects decreased expression of skeletal muscle chloride channel 1 (CLCN1) and the skeletal muscle sodium channel alpha-subunit, resulting in myotonic discharges in skeletal muscle of the lower urinary tract. AR113Q limb muscles show similar myopathic features and express decreased levels of mRNAs encoding neurotrophin-4 and glial cell line-derived neurotrophic factor. These data define an important myopathic contribution to the Kennedy disease phenotype and suggest a role for muscle in non-cell autonomous toxicity of lower motor neurons.     

Hemodynamic response to coupled plasmafiltration-adsorption in human septic shock

OBJECTIVE: The objective was to examine the effect of repeated applications of coupled plasmafiltration-adsorption on the hemodynamic response in septic shock patients hospitalized in intensive care units (ICUs). DESIGN: Prospective, intention-to-treat. SETTING: General ICU of a tertiary care, non-teaching, 400-bed, city hospital. PATIENTS AND PARTICIPANTS: Twelve consecutive mechanically ventilated septic shock patients, with or without concomitant acute renal failure (ARF). INTERVENTION: A median of 10 consecutive sessions (prescribed treatment time: 10 h/session; delivered duration: 8.43+/-1.37 h/min) of coupled plasmafiltration-adsorption for each patient. MEASUREMENTS AND RESULTS: Mean arterial pressure (77.2+/-12.5 [CI 95%; 74.5-79.8] vs. 83.3+/-14.1 [CI 95%; 80.3-86.3] mm Hg; [ p<0.001]), cardiac index (4.03+/-0.89 [CI 95%; 3.83-4.22] vs. 3.46+/-0.82 [CI 95%; 3.28-3.64] L/m(2)/min; [ p<0.001]), systemic vascular resistance index (1,388+/-496 [CI 95%; 1,278-1,497] vs. 1,753+/-516 [CI 95%; 1,639-1,867] dynes x s/cm(5); [ p<0.001]), PO2/FIO2 ratio (204+/-87 [CI 95%; 185-223] vs. 238+/-82 [CI 95%; 220-256]; [ p<0.001]), significantly improved during 100 global treatments (pre- vs. post-treatment values). Intra-thoracic blood volume and extra-vascular lung water did not change across treatments. Vasopressor requirement was reduced: norepinephrine decrease from an infusion rate of 0.13+/-0.07 (CI 95%; 0.06-0.16) to 0 gamma/kg/min after a mean of 5.3+/-2.7 sessions. C reactive protein (CRP) significantly decreased (from 29.3+/-7.3 vs. 7.9+/-4.8; p<0.0001) during treatment. Survival was 90% at day 28 and 70% at day 90. CONCLUSION: Coupled plasmafiltration-adsorption was a feasible and safe extracorporeal treatment and exerted a remarkable improvement in the hemodynamics, the pulmonary function, and the outcome in septic shock patients with or without concomitant ARF.     

Plasma cytokine levels predict response to corticosteroids in septic shock

Purpose

To investigate if plasma cytokine concentrations predict a beneficial response to corticosteroid treatment in septic shock patients.

Methods

A cohort of septic shock patients in whom a panel of 39 cytokines had been measured at baseline (n = 363) was included. Patients who received corticosteroids were propensity score matched to non-corticosteroid-treated patients. An optimal threshold to identify responders to corticosteroid treatment for each cytokine was defined as the concentration above which the odds ratio for 28-day survival between corticosteroid- and non-corticosteroid-treated patients was highest.

Results

Propensity score matching partitioned 165 patients into 61 sets; each set contained matched corticosteroid- and non-corticosteroid-treated patients. For 13 plasma cytokines threshold concentrations were found where the odds ratio for survival between corticosteroid- and non-corticosteroid-treated patients was significant (P < 0.05). CD40 ligand was associated with the highest odds ratio and identified 21 % of the patients in the propensity score matched cohort as responders to corticosteroid treatment. Combinations of triplets of cytokines with a significant odds ratio, using the thresholds identified above, were tested to find a higher proportion of responders. IL3, IL6, and CCL4 identified 50 % of the patients in the propensity score matched cohort as responders to corticosteroid treatment. The odds ratio for 28-day survival was 19 (95 % CI 3.5–140, P = 0.02) with a concentration above threshold for a least one of these cytokines.

Conclusion

Plasma concentration of selected cytokines is a potential predictive biomarker to identify septic shock patients that may benefit from treatment with corticosteroids.

     

Immunologic response to infection and its role in septic shock

In summary, the invasion of bacteria across mucosal surfaces is met with a vigorous host response that includes complement, antibody formation (thymus-independent and eventually thymus-dependent), phagocytosis, production of antibacterial peptides and proteins, the production of cytokines that result in activation of phagocytes and endothelial cells to attract more phagocytes, and the formation of fibrin to limit the spread of infection. The best summary of immune response to infection was written by Lewis Thomas in 1974.     

A dual role for the immune response in a mouse model of inflammation-associated lung cancer

Lung cancer is the leading cause of cancer death worldwide. Both principal factors known to cause lung cancer, cigarette smoke and asbestos, induce pulmonary inflammation, and pulmonary inflammation has recently been implicated in several murine models of lung cancer. To further investigate the role of inflammation in the development of lung cancer, we generated mice with combined loss of IFN-γ and the β-common cytokines GM-CSF and IL-3. These immunodeficient mice develop chronic pulmonary inflammation and lung tumors at a high frequency. Examination of the relationship between these tumors and their inflammatory microenvironment revealed a dual role for the immune system in tumor development. The inflammatory cytokine IL-6 promoted optimal tumor growth, yet wild-type mice rejected transplanted tumors through the induction of adaptive immunity. These findings suggest a model whereby cytokine deficiency leads to oncogenic inflammation that combines with defective antitumor immunity to promote lung tumor formation, representing a unique system for studying the role of the immune system in lung tumor development.     

The contribution of accessory toxins of Vibrio cholerae O1 El Tor to the proinflammatory response in a murine pulmonary cholera model

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.     

Genetic contribution to renal function and electrolyte balance: a twin study

A classical twin study was performed to assess the relative contributions of genetic and environmental factors to serum levels of calcium, phosphate and magnesium, urinary levels of calcium, sodium and potassium, and creatinine clearance. The subjects were 1747 adult female twin pairs: 539 monozygotic and 1208 dizygotic. The intraclass correlations were calculated, and maximum-likelihood model fitting was used to estimate genetic and environmental variance components. The intraclass correlations for all of the variables assessed were higher in monozygotic twin pairs. The heritabilities (with 95% confidence intervals) obtained from model fitting were: serum calcium, 33% (21-45%); serum phosphate, 58% (53-62%), serum magnesium, 27% (15-39%); 24 h urinary potassium, 40% (27-51%); 24 h urinary calcium, 52% (41-61%); 24 h urinary sodium, 43% (30-54%); fractional excretion of sodium, 52% (44-59%); serum creatinine, 37% (25-49); calculated creatinine clearance, 63% (54-72%). This study provides evidence for the importance of genetic factors in determining urinary and blood levels of the major electrolytes involved in blood pressure regulation. Identifying heritability is the first step on the way to finding specific genes, which may improve our insight into the pathophysiology of the metabolism of these electrolytes, and thereby improve our understanding of the aetiology of complex diseases such as renal failure and hypertension.     

Benzodiazepine response and receptor binding after chronic ethanol ingestion in a mouse model.

Benzodiazepine receptor binding and open-field response were examined in male CD-1 mice after 6 weeks on a liquid diet providing either 36% ethanol or maltose-dextrin derived calories. In vivo binding of [3H]Ro15-1788, open-field activity and cortex and plasma concentrations were measured over a range of clonazepam doses (0.05-2.0 mg/kg). Mean +/- S.D. of ethanol consumption was 19.3 +/- 1.1 g/kg/day. Clonazepam concentration in plasma and cortex was related linearly to dose in both groups. Cortex concentrations exceeded plasma concentrations at all doses. Ethanol-consuming mice showed considerably less decrease in measures of horizontal and stereotypic activity at each dose studied. Mean in vivo IC50 was 20.9 +/- 4.0 ng/g for the control mice and 40.2 +/- 7.6 ng/g (P less than .001) in the ethanol-consuming mice. In vitro binding studies found a marked decrease in maximum binding in cortex (37%) with an increase in Kd (66%). Chronic ethanol can influence the acute effects of single doses of clonazepam and both in vivo and in vitro measures of benzodiazepine receptor binding.     

Development of a new model of reconstituted mouse epidermis and characterization of its response to proinflammatory cytokines

The development of three‐dimensional models of reconstituted mouse epidermis (RME) has been hampered by the difficulty to maintain murine primary keratinocyte cultures and to achieve a complete epidermal stratification. In this study, a new protocol is proposed for the rapid and convenient generation of RME, which reproduces accurately the architecture of a normal mouse epidermis. During RME morphogenesis, the expression of differentiation markers such as keratins, loricrin, filaggrin, E‐cadherin and connexins was followed, showing that RME structure at day 5 was similar to those of a normal mouse epidermis, with the acquisition of the natural barrier function. It was also demonstrated that RME responded to skin‐relevant proinflammatory cytokines by increasing the expression of antimicrobial peptides and chemokines, and inhibiting epidermal differentiation markers, as in the human system. This new model of RME is therefore suitable to investigate mouse epidermis physiology further and opens new perspectives to generate reconstituted epidermis from transgenic mice.     

Adrenal response in children with septic shock

Objective To describe the serum cortisol profile and evaluate the adrenal response in children with septic shock, and determine the influence of these factors on the outcome and mortality in this group. Methods Between May and November 2003, 22 children with septic shock admitted to two pediatric intensive care units in southern Brazil were followed. Adrenal function was evaluated based on the levels of cortisol measured on the occasion of the diagnosis of septic shock and on the response of serum cortisol 30 min after the administration of intravenous corticotrophin (0.5 μg/1.73 m²). Adrenal insufficiency was defined as a baseline serum cortisol below 690 nmol/l and/or a cortisol response to corticotrophin less than 250 nmol/l. Results Adrenal insufficiency was detected in 17 patients (77.3%). All patients who died had baseline cortisol higher than 690 nmol/l. A cortisol response to corticotrophin less than 250 nmol/l was associated with a 60% mortality (RR = 7.2, 1.03–50.28). Regression analysis showed that the combination of baseline cortisol higher than 690 nmol/l and a cortisol response to corticotrophin less than 250 nmol/l were associated with mortality after correction for gender and PRISM. Conclusions Adrenal insufficiency is a frequent finding in children with septic shock. The low-dose corticotrophin stimulation test seems to be an important tool to distinguish between a normal cortisol response to stress and evidence of adrenal failure. Mortality was significantly higher in children that failed to respond to a corticotrophin stimulation test.     

Tissue oxygenation and hemodynamic response to NO synthase inhibition in septic shock

The objective of the study was to evaluate the tissue oxygenation and hemodynamic effects of NOS inhibition in clinical severe septic shock. Eight patients with septic shock refractory to volume loading and high level of adrenergic support were prospectively enrolled in the study. Increasing doses of NOS inhibitors [N(G)-nitro-L-arginine-methyl ester (L-NAME) or N(G)-monomethyl-L-arginine (L-NMMA)] were administered as i.v. bolus until a peak effect = 10 mmHg on mean blood pressure was obtained or until side effects occurred. If deemed clinically appropriate, a continuous infusion of L-NAME was instituted and adrenergic support weaning attempted. The bolus administration of NOS inhibitors transiently increased mean blood pressure by 10 mm Hg in all patients. Seven out of eight patients received an L-NAME infusion, associated over 24 h with a progressive decline in cardiac index (P < 0.001) and an increase in systemic vascular resistance (P < 0.01). Partial or total adrenergic support weaning was rapidly possible in 6/8 patients. Oxygen transport decreased (P < 0.001), but oxygen consumption remained unchanged in those patients in whom it could be measured by indirect calorimetry (5/8). Blood lactate and the difference between tonometric gastric and arterial PCO2 remained unchanged. There were 4/8 ICU survivors. We conclude that nitric oxide synthase inhibition in severe septic shock was followed with a progressive correction of the vasoplegic hemodynamic disturbances with finally normalization of cardiac output and systemic vascular resistances without any demonstrable deterioration in tissue oxygenation.     

Inflammatory cytokine response in patients with septic shock secondary to generalized peritonitis

OBJECTIVES: The aims of this study were the following: a) to assess the proinflammatory cytokine (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1, and IL-6) response in patients with septic shock secondary to generalized peritonitis; and b) to evaluate the influence of bacteremic status, type of peritonitis (acute perforation or postoperative), and peritoneal microbial status (mono- or polymicrobial) on cytokine expression and mortality. DESIGN: Prospective study. SETTING: Surgical intensive care unit of a university hospital. PATIENTS: Fifty-two consecutive patients with septic shock caused by generalized peritonitis. INTERVENTIONS: Routine blood tests, blood cultures, and cytokine assays were performed during the first 3 days after onset of shock. MEASUREMENTS AND MAIN RESULTS: Serum TNF-alpha and IL-6 concentrations were measured by using a radioimmunoassay, and IL-1 concentrations were measured by using ELISA. Median serum concentrations on day 1 were: TNF-alpha, 90 pg/mL; IL-1, 7 pg/mL; and IL-6, 5000 pg/mL. TNF-alpha and IL-6 concentrations decreased significantly between the first and third days of septic shock (p = .0001), whereas IL-1 concentrations remained low. The decrease in IL-6 tended to be more pronounced in the survivors group (p = .057). Median TNF-alpha serum concentrations were higher in bacteremic compared with nonbacteremic patients (151 vs. 73 pg/mL, p = .003). TNF-alpha, IL-1, and IL-6 serum concentrations and mortality were not different between acute perforation vs. postoperative peritonitis and mono- versus polymicrobial peritonitis. CONCLUSIONS: The systemic release of TNF-alpha and IL-6 during septic shock caused by generalized peritonitis was maximal on day 1 and decreased rapidly during the next days. No systemic release of IL-1 was observed. IL-6 serum concentrations remained higher in patients who subsequently died. Among the different features of peritonitis studied, only bacteremia influenced the systemic cytokine response (higher TNF-alpha).     

Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.