Protein phosphatase 2A interacts with the Na,K-ATPase and modulates its trafficking by inhibition of its association with arrestin

Background

The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na⁺,K⁺-ATPase. The catalytic subunit of the Na⁺,K⁺-ATPase includes several functional domains that determine its enzymatic and trafficking properties.

Methodology/Principal Findings

Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na⁺,K⁺-ATPase interacting protein. PP-2A C-subunit interacted with the Na⁺,K⁺-ATPase, but not with the homologous sequences of the H⁺,K⁺-ATPase. We confirmed that the Na⁺,K⁺-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na⁺,K⁺-ATPase trafficking through direct association. PP2A inhibits association between the Na⁺,K⁺-ATPase and arrestin, and diminishes the effect of arrestin on Na⁺,K⁺-ATPase trafficking. GRK phosphorylates the Na⁺,K⁺-ATPase and PP2A can at least partially reverse this phosphorylation.

Conclusions/Significance

Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase.     

Metabolic programming during lactation stimulates renal Na+ transport in the adult offspring due to an early impact on local angiotensin II pathways

Background

Several studies have correlated perinatal malnutrition with diseases in adulthood, giving support to the programming hypothesis. In this study, the effects of maternal undernutrition during lactation on renal Na⁺-transporters and on the local angiotensin II (Ang II) signaling cascade in rats were investigated.

Methodology/Principal Findings

Female rats received a hypoproteic diet (8% protein) throughout lactation. Control and programmed offspring consumed a diet containing 20% protein after weaning. Programming caused a decrease in the number of nephrons (35%), in the area of the Bowman''s capsule (30%) and the capillary tuft (30%), and increased collagen deposition in the cortex and medulla (by 175% and 700%, respectively). In programmed rats the expression of (Na⁺+K⁺)ATPase in proximal tubules increased by 40%, but its activity was doubled owing to a threefold increase in affinity for K⁺. Programming doubled the ouabain-insensitive Na⁺-ATPase activity with loss of its physiological response to Ang II, increased the expression of AT₁ and decreased the expression of AT₂ receptors), and caused a pronounced inhibition (90%) of protein kinase C activity with decrease in the expression of the α (24%) and ε (13%) isoforms. Activity and expression of cyclic AMP-dependent protein kinase decreased in the same proportion as the AT₂ receptors (30%). In vivo studies at 60 days revealed an increased glomerular filtration rate (GFR) (70%), increased Na⁺ excretion (80%) and intense proteinuria (increase of 400% in protein excretion). Programmed rats, which had normal arterial pressure at 60 days, became hypertensive by 150 days.

Conclusions/Significance

Maternal protein restriction during lactation results in alterations in GFR, renal Na⁺ handling and in components of the Ang II-linked regulatory pathway of renal Na⁺ reabsorption. At the molecular level, they provide a framework for understanding how metabolic programming of renal mechanisms contributes to the onset of hypertension in adulthood.     

Dose effects of oxaliplatin on persistent and transient Na+ conductances and the development of neurotoxicity

Background

Oxaliplatin, a platinum-based chemotherapy utilised in the treatment of colorectal cancer, produces two forms of neurotoxicity- acute sensorimotor neuropathic symptoms and a dose-limiting chronic sensory neuropathy. Given that a Na⁺ channelopathy has been proposed as the mechanism underlying acute oxaliplatin-induced neuropathy, the present study aimed to determine specific mechanisms of Na⁺ channel dysfunction.

Methodology/Principal Findings

Specifically the function of transient and persistent Na⁺ currents were followed during treatment and were investigated in relation to oxaliplatin dose level. Eighteen patients were assessed before and after a single oxaliplatin infusion with motor and sensory axonal excitability studies performed on the median nerve at the wrist. While refractoriness (associated with Na⁺ channel inactivation) was significantly altered post-oxaliplatin infusion in both motor (Pre: 31.7±6.4%; Post: 68.8±14.5%; P≤.001) and sensory axons (Pre: 31.4±5.4%; Post: 21.4±5.5%; P<.05), strength-duration time constant (marker of persistent Na⁺ conductances) was not significantly altered post-infusion (Motor Pre: 0.395±0.01 ms; Post: 0.394±0.02 ms; NS; Sensory Pre:0.544±0.03 ms; Post: 0.535±0.05 ms; NS). However, changes in strength-duration time constant were significantly correlated with changes in refractoriness in motor and sensory axons (Motor correlation coefficient = −.65; P<.05; Sensory correlation coefficient = .67; P<.05).

Conclusions/Significance

It is concluded that the predominant effect of acute oxaliplatin exposure in human motor and sensory axons is mediated through changes in transient rather than persistent Na⁺ conductances. These findings are likely to have implications for the design and trial of neuroprotective strategies.     

Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues

Background

In contrast to intestinal CD4⁺ regulatory T cells (T_regs), the generation and function of immunomodulatory intestinal CD8⁺ T cells is less well defined. To dissect the immunologic mechanisms of CD8⁺ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.

Methodology and Principal Findings

HA-specific CD8⁺ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3⁺ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8⁺Foxp3⁺ T cells. Antigen-experienced CD8⁺ T cells in this transgenic mouse model suppressed the proliferation of CD8⁺ and CD4⁺ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8⁺ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4⁺ T_reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8⁺Foxp3⁺ T cells.

Conclusion and Significance

We demonstrate that gut specific antigen presentation is sufficient to induce CD8⁺ T_regs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.     

Biased signaling of the angiotensin II type 1 receptor can be mediated through distinct mechanisms

Background

Seven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or β-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of β-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G protein-uncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit β-arrestins. Since uncoupling of G proteins by increased ability to recruit β-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms.

Methodology/Principal Findings

We studied five AT1R mutants previously published to display pathway separation: D74N, DRY/AAY, Y292F, N298A, and Y302F (Ballesteros-Weinstein numbering: 2.50, 3.49–3.51, 7.43, 7.49, and 7.53). We find that D74N, DRY/AAY, and N298A mutants are more prone to β-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit β-arrestin in response to Sar¹-Ile⁴-Ile⁸ (SII) Ang II, a ligand solely activating the β-arrestin pathway.

Conclusions/Significance

Our analysis reveals that the underlying conformations induced by these AT1R mutants most likely represent principally different mechanisms of uncoupling the G protein, which for some mutants may be due to their increased ability to recruit β-arrestin2. Hereby, these findings have important implications for drug discovery and 7TMR biology and illustrate the necessity of uncovering the exact molecular determinants for G protein-coupling and β-arrestin recruitment, respectively.     

Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale

In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).

Objective

To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.

Methods

Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca²⁺ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca²⁺ release, F>F₅₀ within 20 ms) or their absence (delayed areas). Spontaneous Ca²⁺ release events during diastole, Ca²⁺ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.

Results

In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na⁺/Ca²⁺ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca²⁺ influx via Ca²⁺ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca²⁺ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca²⁺ removal by NCX at the membrane was significantly lower in MI.

Conclusion

TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca²⁺ loss and raise SR Ca²⁺ content, but may promote Ca²⁺ waves.     

Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery

Background

The rate-limiting step that determines the dominant time constant (τ_D) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca²⁺-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τ_D is modulated by background light in mouse rods.

Methodology/Principal Findings

We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca²⁺ shortens the dominant time constant (τ_D) of saturated photoresponse recovery, i.e., when extracellular free Ca²⁺ is decreased from 1 mM to ∼25 nM, the τ_D is reversibly increased ∼1.5–2-fold.

Conclusions

We conclude that the increase in τ_D during low Ca²⁺ treatment is not due to increased [cGMP], increased [Na⁺] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca²⁺-dependent mechanism controls the life time of active PDE.     

Circulating CD62E+ microparticles and cardiovascular outcomes

Background

Activated endothelial cells release plasma membrane submicron vesicles expressing CD62E (E-selectin) into blood, known as endothelial microparticles (EMPs). We studied whether the levels of endothelial microparticles expressing CD62E⁺, CD31⁺/Annexin-V⁺, or CD31⁺/CD42⁻ predict cardiovascular outcomes in patients with stroke history.

Methods/Principal Findings

Patients with stroke history at least 3 months prior to enrolment were recruited. Peripheral blood EMP levels were measured by flow cytometry. Major cardiovascular events and death were monitored for 36 months. Three hundred patients were enrolled, of which 298 completed the study according to protocol. Major cardiovascular events occurred in 29 patients (9.7%). Nine patients died, five from cardiovascular causes. Cumulative event-free survival rates were lower in patients with high levels of CD62E⁺ microparticles. Multivariate Cox regression analysis adjusted for cardiovascular risk factors, medications and stroke etiologic groups showed an association between a high CD62E⁺ microparticle level and a risk of major cardiovascular events and hospitalization. Levels of other kinds of EMPs expressing CD31⁺/Annexin-V⁺ or CD31⁺/CD42⁻ markers were not predictive of cardiovascular outcomes.

Conclusion

A high level of CD62E⁺ microparticles is associated with cardiovascular events in patients with stroke history, suggesting that the systemic endothelial activation increases the risk for cardiovascular morbidities.     

A novel and lethal de novo LQT-3 mutation in a newborn with distinct molecular pharmacology and therapeutic response

Background

SCN5A encodes the α-subunit (Na_v1.5) of the principle Na⁺ channel in the human heart. Genetic lesions in SCN5A can cause congenital long QT syndrome (LQTS) variant 3 (LQT-3) in adults by disrupting inactivation of the Na_v1.5 channel. Pharmacological targeting of mutation-altered Na⁺ channels has proven promising in developing a gene-specific therapeutic strategy to manage specifically this LQTS variant. SCN5A mutations that cause similar channel dysfunction may also contribute to sudden infant death syndrome (SIDS) and other arrhythmias in newborns, but the prevalence, impact, and therapeutic management of SCN5A mutations may be distinct in infants compared with adults.

Methods and Results

Here, in a multidisciplinary approach, we report a de novo SCN5A mutation (F1473C) discovered in a newborn presenting with extreme QT prolongation and differential responses to the Na⁺ channel blockers flecainide and mexiletine. Our goal was to determine the Na⁺ channel phenotype caused by this severe mutation and to determine whether distinct effects of different Na⁺ channel blockers on mutant channel activity provide a mechanistic understanding of the distinct therapeutic responsiveness of the mutation carrier. Sequence analysis of the proband revealed the novel missense SCN5A mutation (F1473C) and a common variant in KCNH2 (K897T). Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na⁺ channels revealed significant changes in channel biophysics, all contributing to the proband''s phenotype as predicted by in silico modeling. Furthermore, subtle differences in drug action were detected in correcting mutant channel activity that, together with both the known genetic background and age of the patient, contribute to the distinct therapeutic responses observed clinically.

Significance

The results of our study provide further evidence of the grave vulnerability of newborns to Na⁺ channel defects and suggest that both genetic background and age are particularly important in developing a mutation-specific therapeutic personalized approach to manage disorders in the young.     

Acute lead exposure increases arterial pressure: role of the renin-angiotensin system

Background

Chronic lead exposure causes hypertension and cardiovascular disease. Our purpose was to evaluate the effects of acute exposure to lead on arterial pressure and elucidate the early mechanisms involved in the development of lead-induced hypertension.

Methodology/Principal Findings

Wistar rats were treated with lead acetate (i.v. bolus dose of 320 µg/Kg), and systolic arterial pressure, diastolic arterial pressure and heart rate were measured during 120 min. An increase in arterial pressure was found, and potential roles of the renin-angiotensin system, Na⁺,K⁺-ATPase and the autonomic reflexes in this change in the increase of arterial pressure found were evaluated. In anesthetized rats, lead exposure: 1) produced blood lead levels of 37±1.7 µg/dL, which is below the reference blood concentration (60 µg/dL); 2) increased systolic arterial pressure (Ct: 109±3 mmHg vs Pb: 120±4 mmHg); 3) increased ACE activity (27% compared to Ct) and Na⁺,K⁺-ATPase activity (125% compared to Ct); and 4) did not change the protein expression of the α1-subunit of Na⁺,K⁺-ATPase, AT₁ and AT₂. Pre-treatment with an AT₁ receptor blocker (losartan, 10 mg/Kg) or an ACE inhibitor (enalapril, 5 mg/Kg) blocked the lead-induced increase of arterial pressure. However, a ganglionic blockade (hexamethonium, 20 mg/Kg) did not prevent lead''s hypertensive effect.

Conclusion

Acute exposure to lead below the reference blood concentration increases systolic arterial pressure by increasing angiotensin II levels due to ACE activation. These findings offer further evidence that acute exposure to lead can trigger early mechanisms of hypertension development and might be an environmental risk factor for cardiovascular disease.     

TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8⁺ T cells in both chronic infection and tumor. However, the nature of TIM-3⁺CD4⁺ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4⁺ and CD8⁺ TILs from human lung cancer tissues but negligibly expressed on T cells from patients'' peripheral blood. Frequencies of IFN-γ⁺ cells were reduced in TIM-3⁺CD8⁺ TILs compared to TIM-3⁻CD8⁺ TILs. However, the level of TIM-3 expression on CD8⁺ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3⁺CD4⁺ TILs expressed FOXP3 and about 60% of FOXP3⁺ TILs were TIM-3⁺. Importantly, TIM-3 expression on CD4⁺ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.     

Anti-diarrheal mechanism of the traditional remedy Uzara via reduction of active chloride secretion

Background and Purpose

The root extract of the African Uzara plant is used in traditional medicine as anti-diarrheal drug. It is known to act via inhibition of intestinal motility, but malabsorptive or antisecretory mechanisms are unknown yet.

Experimental Approach

HT-29/B6 cells and human colonic biopsies were studied in Ussing experiments in vitro. Uzara was tested on basal as well as on forskolin- or cholera toxin-induced Cl⁻ secretion by measuring short-circuit current (I_SC) and tracer fluxes of ²²Na⁺ and ³⁶Cl⁻. Para- and transcellular resistances were determined by two-path impedance spectroscopy. Enzymatic activity of the Na⁺/K⁺-ATPase and intracellular cAMP levels (ELISA) were measured.

Key Results

In HT-29/B6 cells, Uzara inhibited forskolin- as well as cholera toxin-induced I_SC within 60 minutes indicating reduced active chloride secretion. Similar results were obtained in human colonic biopsies pre-stimulated with forskolin. In HT-29/B6, the effect of Uzara on the forskolin-induced I_SC was time- and dose-dependent. Analyses of the cellular mechanisms of this Uzara effect revealed inhibition of the Na⁺/K⁺-ATPase, a decrease in forskolin-induced cAMP production and a decrease in paracellular resistance. Tracer flux experiments indicate that the dominant effect is the inhibition of the Na⁺/K⁺-ATPase.

Conclusion and Implications

Uzara exerts anti-diarrheal effects via inhibition of active chloride secretion. This inhibition is mainly due to an inhibition of the Na⁺/K⁺-ATPase and to a lesser extent to a decrease in intracellular cAMP responses and paracellular resistance. The results imply that Uzara is suitable for treating acute secretory diarrhea.     

Different capacity of monocyte subsets to phagocytose iron-oxide nanoparticles

Objective

To explore the capacity of human CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes to phagocyte iron-oxide nanoparticles in vitro.

Methods

Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.

Results

Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14⁺⁺CD16^- monocytes displayed a significantly higher uptake compared to CD14⁺CD16⁺⁺ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes differently.

Conclusion

Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties.     

A functional +61G/A polymorphism in epidermal growth factor is associated with glioma risk among Asians

Background

Epidermal growth factor (EGF), a potent mitogenic protein, plays an important role in the development of cancers, including glioma. Previous studies showed that the EGF +61G/A polymorphism (rs4444903) may lead to an alteration in EGF production and/or activity, which can result in individual susceptibility to glioma. However, published data regarding the association between the +61G/A polymorphism and glioma risk was contradictory.

Objective

The aim of this study was to perform a meta-analysis of eligible studies to derive precise estimation of the association of EGF +61G/A with glioma risk.

Methods

We performed a pooled analysis of seven published studies that included 1,613 glioma cases and 2,267 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association. The pooled ORs were performed for codominant model, dominant model, and recessive model, respectively.

Results

Overall, no significant associations between the EGF +61G/A polymorphism and glioma cancer risk were found for AA versus GG (OR = 0.95, 95% CI = 0.62–1.45), GA versus GG (OR = 0.94, 95% CI = 0.72–1.22), AA/GA versus GG (OR = 0.93, 95% CI = 0.70–1.23), and AA versus GA/GG (OR = 1.04, 95% CI = 0.77–1.39). However, in the stratified analysis by ethnicity, the EGF +61G/A polymorphism had a higher risk of glioma development among Asians, but a lower risk among Caucasians.

Conclusions

Taken together, the results suggest that the EGF +61G/A polymorphism may contribute to the susceptibility of glioma in different ethnic groups.     

The type of responder T-cell has a significant impact in a human in vitro suppression assay

Background

In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4⁺CD25^+high, or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4⁺CD25^low T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease.

Methods/Principal Findings

We investigated human CD4⁺CD25^low T cells and compared them to CD4⁺CD25^- T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4⁺CD25^low T cells divided more rapidly than CD4⁺CD25^- T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25^low compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25^low T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively).

Conclusions/Significance

The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases.     

In vivo determination of mouse olfactory mucus cation concentrations in normal and inflammatory States

Objective

Olfaction is impaired in chronic rhinosinusitis (CRS). The study has two aims: (1) to determine whether changes in cation concentration occur in the olfactory mucus of mice with CRS, which may affect chemo-electrical transduction, (2) and to examine whether these alterations are physiologically significant in humans.

Study Design

Animal study in mice and translational study in humans.

Methods

Inflammation was induced by sensitization and chronic exposure of 16 C57BL/6 mice to Aspergillus fumigatus. The control group included 16 untreated mice. Ion-selective microelectrodes were used to measure free cation concentrations in the olfactory mucus of 8 mice from each treatment group, while the remaining mice were sacrificed for histology. To validate the findings in the animal model, olfactory threshold was measured in 11 healthy human participants using Sniffin’ Sticks before and after nasal irrigation with solutions that were composed of either of the cation concentrations.

Results

In 8 mice, olfactory mucus of chronically inflamed mice had lower [Na⁺] (84.8±4.45 mM versus 93.73±3.06 mM, p = 0.02), and higher [K⁺] (7.2±0.65 mM versus 5.7±0.20 mM, p = 0.04) than controls. No difference existed in [Ca²⁺] (0.50±0.12 mM versus 0.54±0.06 mM, p = 0.39). In humans, rinsing with solutions replicating ion concentrations of the mouse mucosa with chronic inflammation caused a significant elevation in the median olfactory threshold (9.0 to 4.8, p = 0.003) but not with the control solution (8.3 to 7.8, p = 0.75).

Conclusion

Chronic inflammation elevates potassium and lowers sodium ion concentration in mice olfactory mucus. Nasal irrigation with a corresponding solution induced olfactory threshold shift in humans.     

Surveillance for Clostridium difficile infection: ICD-9 coding has poor sensitivity compared to laboratory diagnosis in hospital patients, Singapore

Introduction

Clostridium difficile infection (CDI) is an increasingly recognized nosocomial infection in Singapore. Surveillance methods include laboratory reporting of Clostridium difficile toxin assays (CDTA) or use of International Classification of Diseases, 9^th Revision (ICD-9) discharge code 008.45. Previous US studies showed good correlation between CDTA and ICD-9 codes. However, the use of ICD-9 codes for CDI surveillance has not been validated in other healthcare settings.

Methods

We compared CDI rates based on CDTA to ICD-9 codes for all discharges in 2007 from our hospital to determine sensitivity and specificity of ICD-9 codes. Demographic and hospitalization data were analyzed to determine predictors for missing ICD-9 codes.

Results

During 2007, there were 56,352 discharges. Of these, 268 tested CDTA-positive but only 133 were assigned the CDI ICD-9 code. A total of 141 discharges had the ICD-9 code; 8 were CDTA-negative, the rest were CDTA-positive. Community-acquired CDI accounted for only 3.2% of cases. The sensitivity and specificity of ICD-9 codes compared to CDTA were 49.6% and 100% respectively. Concordance between CDTA and ICD-9 codes was 0.649 (p<.001). Comparing concordant patients (CDTA+/ICD9+) to discordant patients (CDTA+/ICD9−), concordant patients were more likely to be over 50 years of age (OR 3.49, 95% CI 1.66–7.34, p = .001) and have shorter time from admission to testing (OR 0.98, 95% CI 0.97–0.99, p = .009).

Discussion

Unlike previous studies in the US, ICD-9 codes substantially underestimate CDI in Singapore compared to microbiological data. Older patients with shorter time to testing were less likely to have missing ICD-9 codes.     

HIV-specific T-cells accumulate in the liver in HCV/HIV co-infection

Background and Aims

Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses.

Methods

Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry.

Results

HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4⁺ T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8⁺ and CD4⁺ T-cells producing IFN-γ and TNF-α were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8⁺ T-cells produced more of the fibrogenic cytokine, TNF-α. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4⁺ T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses.

Conclusion

The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease.