Electrical Impedance Myography to Detect the Effects of Electrical Muscle Stimulation in Wild Type and Mdx Mice

Objective

Tools to better evaluate the impact of therapy on nerve and muscle disease are needed. Electrical impedance myography (EIM) is sensitive to neuromuscular disease progression as well as to therapeutic interventions including myostatin inhibition and antisense oligonucleotide-based treatments. Whether the technique identifies the impact of electrical muscle stimulation (EMS) is unknown.

Methods

Ten wild-type (wt) C57B6 mice and 10 dystrophin-deficient (mdx) mice underwent 2 weeks of 20 min/day EMS on left gastrocnemius and sham stimulation on the right gastrocnemius. Multifrequency EIM data and limb girth were obtained before and at the conclusion of the protocol. Muscle weight, in situ force measurements, and muscle fiber histology were also assessed at the conclusion of the study.

Results

At the time of sacrifice, muscle weight was greater on the EMS-treated side than on the sham-stimulated side (p = 0.018 for wt and p = 0.007 for mdx). Similarly, in wt animals, EIM parameters changed significantly compared to baseline (resistance (p = 0.009), reactance (p = 0.0003) and phase (p = 0.002); these changes were due in part to reductions in the EIM values on the EMS-treated side and elevations on the sham-simulated side. Mdx animals showed analogous but non-significant changes (p = 0.083, p = 0.064, and p = 0.57 for resistance, reactance and phase, respectively). Maximal isometric force trended higher on the stimulated side in wt animals only (p = 0.06). Myofiber sizes in wt animals were also larger on the stimulated side than on the sham-stimulated side (p = 0.034); no significant difference was found in the mdx mice (p = 0.79).

Conclusion

EIM is sensitive to stimulation-induced muscle alterations in wt animals; similar trends are also present in mdx mice. The mechanisms by which these EIM changes develop, however, remains uncertain. Possible explanations include longer-term trophic effects and shorter-term osmotic effects.     

Renal Function Trajectories in Patients with Prior Improved eGFR Slopes and Risk of Death

Background

Multiple prior studies demonstrated that patients with early Chronic Kidney Disease (CKD) and positive estimated Glomerular Filtration Rate (eGFR) slopes experience increased risk of death. We sought to characterize patients with positive eGFR slopes, examine the renal function trajectory that follows the time period where positive slope is observed, and examine the association between different trajectories and risk of death.

Methods and Findings

We built a cohort of 204,132 United States veterans with early CKD stage 3; eGFR slopes were defined based on Bayesian mixed-effects models using outpatient eGFR measurements between October 1999 and September 2004; to build renal function trajectories, patients were followed longitudinally thereafter (from October 2004) until September 2013. There were 41,410 (20.29%) patients with positive eGFR slope and they exhibited increased risk of death compared to patients with stable eGFR slope (HR = 1.33, CI:1.31–1.35). There was an inverse graded association between severity of albuminuria and the odds of positive eGFR slope (OR = 0.94, CI:0.90–0.98, and OR = 0.76, CI:0.69–0.84 for microalbuminuria and albuminuria; respectively). Following the time period where positive eGFR slope is observed, we characterized 4 trajectory phenotypes: high eGFR intercept and positive trajectory (HIPT) (12.42%), intermediate intercept and mild negative trajectory (IIMNT) (60.04%), low intercept and fast negative trajectory (LIFNT)(23.33%), and high intercept and fast negative trajectory (HIFNT) (4.20%). Compared to IIMNT (reference group), HIPT is associated with younger age, dementia, HIV, chronic lung disease, peripheral artery disease, weight loss, and inversely associated with albuminuria; LIFNT and HIFNT were associated with diabetes, hypertension, cardiovascular disease, peripheral artery disease, and albuminuria. The risk of death at 9 years was lowest in IIMNT (HR = 1.12, CI:1.09–1.14), highest in HIPT (HR = 1.71, CI:1.63–1.79), and intermediate in LIFNT (HR = 1.36, CI:1.32–1.40) and HIFNT (HR = 1.56, CI:1.45–1.68).

Conclusions

Our results demonstrate that patients with positive eGFR slopes, when followed over longer period of time, follow 4 distinct trajectory phenotypes that have distinct demographic and clinical correlates and are differentially associated with risk of death.     

Alterations in Cortical Sensorimotor Connectivity following Complete Cervical Spinal Cord Injury: A Prospective Resting-State fMRI Study

Functional magnetic resonance imaging (fMRI) studies have demonstrated alterations during task-induced brain activation in spinal cord injury (SCI) patients. The interruption to structural integrity of the spinal cord and the resultant disrupted flow of bidirectional communication between the brain and the spinal cord might contribute to the observed dynamic reorganization (neural plasticity). However, the effect of SCI on brain resting-state connectivity patterns remains unclear. We undertook a prospective resting-state fMRI (rs-fMRI) study to explore changes to cortical activation patterns following SCI. With institutional review board approval, rs-fMRI data was obtained in eleven patients with complete cervical SCI (>2 years post injury) and nine age-matched controls. The data was processed using the Analysis of Functional Neuroimages software. Region of interest (ROI) based analysis was performed to study changes in the sensorimotor network using pre- and post-central gyri as seed regions. Two-sampled t-test was carried out to check for significant differences between the two groups. SCI patients showed decreased functional connectivity in motor and sensory cortical regions when compared to controls. The decrease was noted in ipsilateral, contralateral, and interhemispheric regions for left and right precentral ROIs. Additionally, the left postcentral ROI demonstrated increased connectivity with the thalamus bilaterally in SCI patients. Our results suggest that cortical activation patterns in the sensorimotor network undergo dynamic reorganization following SCI. The presence of these changes in chronic spinal cord injury patients is suggestive of the inherent neural plasticity within the central nervous system.     

The Gap between Individual Perception and Compliance: A Qualitative Follow-Up Study of the Surgical Safety Checklist Application

Background

“The Surgical Safety Checklist (SSC) is important, but we don’t use it adequately” is a well-suited statement that reflects the SSC''s application in hospitals. Our aim was to follow up on our initial study on compliance (2014) by analysing differences between individual perception and compliance with the SSC.

Methods

We conducted a follow-up online survey to assess healthcare professionals’ individual perception of, as well as satisfaction and compliance with the SSC three years following its thorough implementation.

Results

171 (19.5%) of 875 operating team members completed the online survey. 99.4% confirmed using the SSC. Self-estimated subjective knowledge about the intention of the checklist was high, whereas objective knowledge was moderate, but improved as compared to 2014. According to an independent audit the SSC was used in 93.1% of all operations and among the SSCs used the completion rate was 57.2%. The use of the SSC was rated as rather easy [median (IQR): 7 (6–7)], familiar [7 (6–7)], generally important [7 (7–7)], and good for patients [7 (6–7)] as well as for employees [7 (7–7)]. Only comfort of use was rated lower [6 (5–7)].

Conclusion

There is a gap between individual perception and actual application of the SSC. Despite healthcare professionals confirming the importance of the SSC, compliance was moderate. The introduction of SSCs in the health care sector remains a constant challenge and requires continuous re-evaluation as well as a sensible integration into existing workflows in hospitals.     

Strengthening Community-Based Vital Events Reporting for Real-Time Monitoring of Under-Five Mortality: Lessons Learned from the Balaka and Salima Districts in Malawi

Background

Malawi ratified a compulsory birth and death registration system in 2009. Until it captures complete coverage of vital events, Malawi relies on other data sources to calculate mortality estimates. We tested a community-based method to estimate annual under-five mortality rates (U5MR) through the Real-Time Monitoring of Under-Five Mortality (RMM) project in Malawi. We implemented RMM in two phases, and conducted an independent evaluation of phase one after 21 months of implementation. We present results of the phase two validation that covers the full project time span, and compare the results to those of the phase one validation.

Methods and Findings

We assessed the completeness of the counts of births and deaths and the accuracy of disaggregated U5MR from the community-based method against a retrospective full pregnancy history for rolling twelve-month periods after the independent evaluation. We used full pregnancy histories collected through household interviews carried out between November 2013 and January 2014 as the validation data source. Health Surveillance Agents (HSAs) across the 160 catchment areas submitted routine reports on pregnancies, births, and deaths consistently. However, for the 15-month implementation period post-evaluation, average completeness of birth event reporting was 76%, whereas average completeness of death event reporting was 67% relative to that expected from a comparable pregnancy history. HSAs underestimated the U5MR by an average of 21% relative to that estimated from a comparable pregnancy history.

Conclusions

On a medium scale, the community-based RMM method in Malawi produced substantial underestimates of annualized U5MR relative to those obtained from a full pregnancy history, despite the additional incentives and quality-control activities. We were not able to achieve an optimum level of incentive and support to make the system work while ensuring sustainability. Lessons learned from the implementation of RMM can inform programs supporting community-based interventions through HSAs in Malawi.     

Proteomic changes in response to crystal formation in Drosophila Malpighian tubules

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.     

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.     

Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE)

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl₂ differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.     

Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

BackgroundCD19⁺CD24^hiCD38^hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus.MethodsPBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA.ResultsThe amount of CD19⁺ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%.ConclusionCalcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.