Characterising plasmacytoid and myeloid AXL+ SIGLEC-6+ dendritic cell functions and their interactions with HIV.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.

Four-limb wireless IMU sensor system for automatic gait detection in canines.

This study aims to develop a 4-limb canine gait analysis system using wireless inertial measurement units (IMUs). 3D printed sensor holders were designed to ensure quick and consistent sensor mounting. Signal analysis algorithms were developed to automatically determine the timing of swing start and end in a stride. To evaluate the accuracy of the new system, a synchronized study was conducted in which stride parameters in four dogs were measured simultaneously using the 4-limb IMU system and a pressure-sensor based walkway gait system. The results showed that stride parameters measured in both systems were highly correlated. Bland-Altman analyses revealed a nominal mean measurement bias between the two systems in both forelimbs and hindlimbs. Overall, the disagreement between the two systems was less than 10% of the mean value in over 92% of the data points acquired from forelimbs. The same performance was observed in hindlimbs except for one parameter due to small mean values. We demonstrated that this 4-limb system could successfully visualize the overall gait types and identify rapid gait changes in dogs. This method provides an effective, low-cost tool for gait studies in veterinary applications or in translational studies using dog models of neuromuscular diseases.

Extensor carpi ulnaris muscle shows unexpected slow-to-fast fiber-type switch in Duchenne muscular dystrophy dogs.

Aged dystrophin-null canines are excellent models for studying experimental therapies for Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. To establish the baseline, we studied the extensor carpi ulnaris (ECU) muscle in 15 terminal age (3-year-old) male affected dogs and 15 age/sex-matched normal dogs. Affected dogs showed histological and anatomical hallmarks of dystrophy, including muscle inflammation and fibrosis, myofiber size variation and centralized myonuclei, as well as a significant reduction of muscle weight, muscle-to-body weight ratio and muscle cross-sectional area. To rigorously characterize the contractile properties of the ECU muscle, we developed a novel in situ assay. Twitch and tetanic force, contraction and relaxation rate, and resistance to eccentric contraction-induced force loss were significantly decreased in affected dogs. Intriguingly, the time-to-peak tension and half-relaxation time were significantly shortened in affected dogs. Contractile kinetics predicted an unforeseen slow-to-fast myofiber-type switch, which we confirmed at the protein and transcript level. Our study establishes a foundation for studying long-term and late-stage therapeutic interventions in dystrophic canines. The unexpected myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals. This article has an associated First Person interview with the first author of the paper.

Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

An improved method for studying mouse diaphragm function.

Dysfunction in the contractile properties of the diaphragm muscle contributes to the morbidity and mortality in many neuromuscular and respiratory diseases. Methods that can accurately quantify diaphragm function in mouse models are essential for preclinical studies. Diaphragm function is usually measured using the diaphragm strip. Two methods have been used to attach the diaphragm strip to the force transducer. The suture method is easy to adopt but it cannot maintain the physiological orientation of the muscle fibers. Hence, results may not accurately reflect diaphragm contractility. The clamp method can better maintain diaphragm muscle fiber orientation but is used less often because detailed information on clamp fabrication and application has never been published. Importantly, a side-by-side comparison of the two methods is lacking. To address these questions, we engineered diaphragm clamps using mechanically highly durable material. Here, we present a detailed and ready-to-use protocol on the design and manufacture of diaphragm clamps. Also, we present a step by step protocol on how to mount the diaphragm strip to the clamp and then to the muscle force measurement system. We compared the diaphragm force from the same mouse with both suture and clamp methods. We found the clamp method yielded a significantly higher muscle force. Finally, we validated the utility of the clamp method in the mdx model of Duchenne muscular dystrophy. In summary, the clamp method described in this paper yields reliable and consistent diaphragm force data. This method will be useful to any laboratory interested in performing mouse diaphragm function assay.

Automatic characterization of stride parameters in canines with a single wearable inertial sensor.

BACKGROUND AND OBJECTIVE: Gait analysis is valuable for studying neuromuscular and skeletal diseases. Wearable motion sensors or inertial measurement units (IMUs) have become common for human gait analysis. Canines are important large animal models for translational research of human diseases. Our objective is to develop a method for accurate and reliable determination of the timing of each stride in dogs using a wearable IMU. METHODS: We built a wireless IMU sensor using off-the-shelf components. We also developed a MATLAB algorithm for data acquisition and stride timing determination. Stride parameters from 1,259 steps of three adult mixed breed dogs were determined across a range of six height-normalized speeds using the IMU system. The IMU results were validated by frame-by-frame manual counting of high-speed video recordings. RESULTS: Comparing IMU derived results with video revealed that the mean error ± standard deviation for stride, stance, and swing duration was 0.001 ± 0.025, -0.001 ± 0.030, and 0.001 ± 0.019 s respectively. A mean error ± standard deviation of 0.000 ± 0.020 and -0.008 ± 0.027 s was obtained for determining toe-off and toe-touch events respectively. Only one step was missed by the algorithm in the video dataset of 1,259 steps. CONCLUSION: We have developed and validated an IMU method for automatic canine gait analysis. Our method can be used for studying neuromuscular diseases in veterinary clinics and in translational research.

AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice.

Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.

Detecting Associations between Major Depressive Disorder Treatment and Essential Hypertension using Electronic Health Records.

In this observational study, we investigate the correlation between depression and hypertension on a cohort of patients treated for major depressive disorder using Selective Serotonin Reuptake Inhibitors (SSRIs) and assess the effect of depression treatment on the diagnoses and treatment for essential hypertension. Our results indicate that the positive effect of successful depression treatment can be discovered and estimated from electronic health record (EHR) data even for a small sample size. We have also successfully detected differences in the effect of depression treatment in hypertensive patients between the two phenotypes representing successful treatment outcomes-response and remission- concluding that achieving remission has a longer lasting effect than response.