Acoustic radiation force impulse shear wave elastography quantifies upper limb muscle in patients with Duchenne muscular dystrophy.

We investigated whether the upper limb muscle stiffness quantified by the acoustic radiation force impulse shear wave elastography (ARFI/SWE) is a potential biomarker for age-related muscle alteration and functional decline in patients with Duchenne muscular dystrophy (DMD). 37 patients with DMD and 30 typically developing controls (TDC) were grouped by age (3-8, 9-11, and 12-18 years). ARFI/SWE measured the biceps and deltoid muscle's shear wave velocities (SWVs). Performance of Upper Limb Module (PUL 1.2 module) assessed muscle function in DMD patients. Mann Whitney test compared muscle SWVs between DMD and TDC, stratified by three age groups. We used analysis of variance with Bonferroni correction to compare muscle SWVs between DMD and TDC and correlated muscle SWVs with PUL results in the DMD group. Results showed that the SWVs of biceps differentiated DMD patients from TDC across age groups. Younger DMD patients (3-8 years) exhibited higher SWVs (p = 0.013), but older DMD patients (12-18 years) showed lower SWVS (p = 0.028) than same-aged TDC. DMD patients had decreasing biceps SWVs with age (p < 0.001), with no such age effect in TDC. The SWVs of deltoid and biceps positively correlated with PUL scores (r = 0.527 âˆ¼ 0.897, P < 0.05) and negatively correlated with PUL timed measures (r = -0.425 âˆ¼ -0.542, P < 0.05) in DMD patients. Our findings suggest that ARFI/SWE quantifying the SWVs in upper limb muscle could be a potential biomarker to differentiate DMD from TDC across ages and that DMD patients showed age-related muscle alteration and limb functional decline.

PD-L1 Enhanced by cis-Urocanic Acid on Langerhans Cells Inhibits Vγ4+ γδT17 Cells in Imiquimod-Induced Skin Inflammation.

Psoriasis is an IL-23/IL-17-mediated inflammatory autoimmune dermatosis, and UVB may contribute to immunosuppression and ameliorate associated symptoms. One of the pathophysiology underlying UVB therapy is the production of cis-urocanic acid (cis-UCA) by keratinocytes. However, the detailed mechanism is yet to be fully understood. In this study, we found FLG expression and serum cis-UCA levels were significantly lower in patients with psoriasis than in healthy controls. We also noted that cis-UCA application inhibited psoriasiform inflammation through the reduction of Vγ4+ γδT17 cells in murine skin and draining lymph nodes. Meanwhile, CCR6 was downregulated on γδT17 cells, which would suppress the inflammatory reaction at a distal skin site. We revealed that the 5-hydroxytryptamine receptor 2A, the known cis-UCA receptor, was highly expressed on Langerhans cells in the skin. cis-UCA also inhibited IL-23 expression and induced PD-L1 on Langerhans cells, leading to the attenuated proliferation and migration of γδT-cells. Compared to the isotype control, α-PD-L1 treatment in vivo could reverse the antipsoriatic effects of cis-UCA. PD-L1 expression on Langerhans cells was sustained through the cis-UCA-induced mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. These findings uncover the cis-UCA-induced PD-L1-mediated immunosuppression on Langerhans cells, which facilitates the resolution of inflammatory dermatoses.

Quantifying Lower Limb Muscle Stiffness as Ambulation Function Declines in Duchenne Muscular Dystrophy with Acoustic Radiation Force Impulse Shear Wave Elastography.

Duchenne muscular dystrophy (DMD) is a progressive muscular disease, but validated imaging tools to quantify muscle microstructure alteration as mobility declines are lacking. We aimed to determine the feasibility of using acoustic radiation force impulse shear-wave elastography (ARFI/SWE) in the quantitative assessment of lower limb muscle stiffness in DMD patients. Shear wave velocities (SWVs) of lower limbs were measured in 39 DMD patients and 36 healthy controls aged 3-20 y. Mean SWV values of the controls and of the DMD patients at different ambulatory stages were compared using analysis of variance with Bonferroni correction. The DMD group had increased lower limb muscle stiffness compared with controls. Stiffness of the tibialis anterior and medial gastrocnemius muscle decreased from ambulatory to early non-ambulatory stages, whereas stiffness of the rectus femoris muscle increased from ambulatory to late non-ambulatory stages. We describe how SWV changes in lower limb muscles have the potential to predict ambulatory decline in DMD.

Deep Learning of Ultrasound Imaging for Evaluating Ambulatory Function of Individuals with Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) results in loss of ambulation and premature death. Ultrasound provides real-time, safe, and cost-effective routine examinations. Deep learning allows the automatic generation of useful features for classification. This study utilized deep learning of ultrasound imaging for classifying patients with DMD based on their ambulatory function. A total of 85 individuals (including ambulatory and nonambulatory subjects) underwent ultrasound examinations of the gastrocnemius for deep learning of image data using LeNet, AlexNet, VGG-16, VGG-16TL, VGG-19, and VGG-19TL models (the notation TL indicates fine-tuning pretrained models). Gradient-weighted class activation mapping (Grad-CAM) was used to visualize features recognized by the models. The classification performance was evaluated using the confusion matrix and receiver operating characteristic (ROC) curve analysis. The results show that each deep learning model endows muscle ultrasound imaging with the ability to enable DMD evaluations. The Grad-CAMs indicated that boundary visibility, muscular texture clarity, and posterior shadowing are relevant sonographic features recognized by the models for evaluating ambulatory function. Of the proposed models, VGG-19 provided satisfying classification performance (the area under the ROC curve: 0.98; accuracy: 94.18%) and feature recognition in terms of physical characteristics. Deep learning of muscle ultrasound is a potential strategy for DMD characterization.

Blocking pannexin1 reduces airway inflammation in a murine model of asthma.

Stressed or injured cells release ATP into the extracellular milieu via the pannexin1 (Panx1) channels, which is the basis of inflammation in a variety of conditions, including allergic lung inflammation. Although the role of Panx1 in mediating inflammation has been well established, the role of its mimetic peptide, 10Panx1, which inhibits ATP release from Panx1 channels, in allergic asthma remains understudied. The aim of this study was to evaluate the effects of using 10Panx1 to inhibit Panx1 channel in a murine model of ovalbumin (OVA)-induced asthma. We demonstrate that blockade of Panx1 significantly attenuated goblet cell hyperplasia and inflammatory cell infiltration into the lungs of OVA-sensitized mice. Inhibition of Panx1 also reduced the total and eosinophil cell numbers in the bronchoalveolar lavage fluid (BALF) and reduced expression of CCL11 and CCL2 in lung tissues from mice. Moreover, we detected lower levels of IL-5 and IL-13 in the culture supernatant of OVA-restimulated splenocytes from 10Panx1-treated mice. Collectively, our findings suggest that Panx1 inhibition of allergen-mediated lung inflammation has the potential to suppress allergic responses in asthma.

DNA methyltransferase inhibitor alleviates bleomycin-induced pulmonary inflammation.

Acute lung injury (ALI) is a severe disease characterized by several inflammatory responses in the lung with high mortality. We applied a mouse model of the pulmonary inflammation induced by the intratracheal instillation of bleomycin which is widely used to induce ALI and fibrosis in animal models. We hypothesized that DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), with its anti-inflammatory benefits, might attenuate bleomycin-induced ALI through the alleviation of inflammation in the lung. We quantified white blood cells with cell blood count (CBC) test, lung inflammation by analyzing cells in the collected bronchoalveolar lavage fluid (BALF) and histological analysis of the lung tissues, and gene expression levels by real-time PCR. Intratracheal administration of bleomycin in mice induced pulmonary inflammation, characterized by increased neutrophil infiltration and inflammatory cytokine expression in the lungs. Mice treated with 5-Aza showed a significant reduction of lung neutrophilia, together with lower expressions of CXCL2 and MCP-1. Furthermore, 5-Aza treatment decreased the expression of proinflammatory cytokines in the lung tissue. Collectively, our data show that DNA methyltransferase inhibitor can alleviate the lung inflammation of bleomycin-induced ALI, indicating an alternative treatment option for the inflammation-triggered lung injury.

Metabolic adaptation orchestrates tissue context-dependent behavior in regulatory T cells.

The diverse distribution and functions of regulatory T cells (Tregs) ensure tissue and immune homeostasis; however, it remains unclear which factors can guide distribution, local differentiation, and tissue context-specific behavior in Tregs. Although the emerging concept that Tregs could re-adjust their transcriptome based on their habitations is supported by recent findings, the underlying mechanisms that reprogram transcriptome in Tregs are unknown. In the past decade, metabolic machineries have been revealed as a new regulatory circuit, known as immunometabolic regulation, to orchestrate activation, differentiation, and functions in a variety of immune cells, including Tregs. Given that systemic and local alterations of nutrient availability and metabolite profile associate with perturbation of Treg abundance and functions, it highlights that immunometabolic regulation may be one of the mechanisms that orchestrate tissue context-specific regulation in Tregs. The understanding on how metabolic program instructs Tregs in peripheral tissues not only represents a critical opportunity to delineate a new avenue in Treg biology but also provides a unique window to harness Treg-targeting approaches for treating cancer and autoimmunity with minimizing side effects. This review will highlight the metabolic features on guiding Treg formation and function in a disease-oriented perspective and aim to pave the foundation for future studies.

MiR-23~27~24-mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation.

Follicular helper T (TFH) cells are essential for generating protective humoral immunity. To date, microRNAs (miRNAs) have emerged as important players in regulating TFH cell biology. Here, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated TFH cell frequencies upon different immune challenges, whereas overexpression of this miRNA family led to reduced TFH cell responses. Mechanistically, miR-23~27~24 clusters coordinately control TFH cells through targeting a network of genes that are crucial for TFH cell biology. Among them, thymocyte selection-associated HMG-box protein (TOX) was identified as a central transcription regulator in TFH cell development. TOX is highly up-regulated in both mouse and human TFH cells in a BCL6-dependent manner. In turn, TOX promotes the expression of multiple molecules that play critical roles in TFH cell differentiation and function. Collectively, our results establish a key miRNA regulon that maintains optimal TFH cell responses for resultant humoral immunity.

A Novel CD3G Mutation in a Taiwanese Patient With Normal T Regulatory Function Presenting With the CVID Phenotype Free of Autoimmunity-Analysis of all Genotypes and Phenotypes.

The T-cell receptor (TCR)/CD3 complex is crucial for T-cell development and regulation. In humans, CD3D, CD3E, and CD3Z gene defects cause severe combined T- and B-cell immunodeficiency. However, CD3G mutations alone lead to a less severe condition, which is mainly characterized by autoimmunity. In the present study, we report the case of a 36-year-old male who presented with recurrent sinopulmonary infections without opportunistic infections; this was compatible with hypogammaglobulinemia, but normal PHA-lymphocyte proliferation. This patient had the common variable immunodeficiency (CVID) phenotype and received regular immunoglobulin infusions over 20-years; he gradually developed nodular regenerative hyperplasia over a 5-year period. Distinct from the previously reported CD3G mutations, which mainly present as autoimmunity, the novel CD3G deletion (c.del213A) in our patient caused an obvious decrease in switched memory B cells and diminished CD40L expression. However, sufficient Treg suppression function was maintained so that he remained free of autoimmune thyroiditis (AIT), inflammatory bowel disease (IBD), and autoimmune pancytopenia. A PubMed search for this rare disease entity revealed seven Turkish and two Spanish patients (five unrelated families). Among a total of 20 alleles, there were 14 splicing mutations (80(-1)G>C), two missense mutations (c.1G>A), two nonsense mutations (c.250A>T), and two deletions (c.del213A). Three patients presented with isolated AIT without significant infections. Three patients died, one from a severe infection at 31 months, one from post-transplant respiratory failure due to viral pneumonia at 17 months, and one from graft-vs.-host disease at 47 months. Those experiencing opportunistic infections, severe life-threatening infections in need of hematopoietic stem cell transplantation, and IBD-like diarrhea had a significantly higher mortality rate compared with those without these features (p = 0.0124, p = 0.01, and p = 0.0124, respectively). The patients with AIT had a significantly better prognosis (p = 0.0124) to those without AIT. Our patient with the novel CD3G mutation presented with predominant B-cell deficiency overlapping with the CVID phenotype but without recognizable autoimmunity, which was consistent with his normal Treg suppression function.

Evaluation of muscular changes by ultrasound Nakagami imaging in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common debilitating muscular disorder. Developing a noninvasive measure for monitoring the progression of this disease is critical. The present study tested the effectiveness of using ultrasound Nakagami imaging to evaluate the severity of the dystrophic process. A total of 47 participants (40 with DMD and 7 healthy controls) were recruited. Patients were classified into stage 1 (presymptomatic and ambulatory), stage 2 (early nonambulatory), and stage 3 (late nonambulatory). All participants underwent ultrasound examinations on the rectus femoris, tibialis anterior, and gastrocnemius. The results revealed that the ultrasound Nakagami parameter correlated positively with functional severity in the patients with DMD. The median Nakagami parameter of the gastrocnemius muscle increased from 0.50 to 0.85, corresponding to the largest dynamic range between normal and stage 3. The accuracy, sensitivity, and specificity of diagnosing walking function were 85.52%, 76.31%, and 94.73%, respectively. The Nakagami parameter of the rectus femoris and gastrocnemius muscles correlated negatively with the 6-minute walking distance in the ambulatory patients. Therefore, changes in the Nakagami parameter for the gastrocnemius muscle are suitable for monitoring disease progression in ambulatory patients and for predicting ambulation loss. Ultrasound Nakagami imaging shows potential for evaluating patients with DMD.

DNA Methyltransferase Inhibitor Promotes Human CD4+CD25hFOXP3+ Regulatory T Lymphocyte Induction under Suboptimal TCR Stimulation.

The "master transcription factor" FOXP3 regulates the differentiation, homeostasis, and suppressor function of CD4+ regulatory T (Treg) cells, which are critical in maintaining immune tolerance. Epigenetic regulation of FOXP3 expression has been demonstrated to be important to Treg cell development, but the induction of human Treg cells through epigenetic modification has not been clearly described. We report that the combination of the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and suboptimal T cell receptor (TCR) stimulation promoted CD4+CD25hFOXP3+ T cell induction from human CD4+CD25- T cells. 5-Aza treatment enhanced the expression of Treg cell signature genes, such as CD25, FOXP3, CTLA-4, and GITR, in CD4+CD25h cells. Moreover, 5-Aza-treated CD4+CD25h T cells showed potent suppressive activity in a cell contact-dependent manner and reduced methylation in the Treg-specific demethylated region (TSDR) in the FOXP3 gene. The analysis of cytokine production revealed that CD4+CD25- T cells with 5-Aza treatment produced comparable levels of interferon (IFN)-γ and transforming growth factor (TGF)-ß, but less IL-10 and more IL-2, when compared to cells without 5-Aza treatment. The increased IL-2 was indispensible to the enhanced FOXP3 expression in 5-Aza-treated CD4+CD25h cells. Finally, 5-Aza-treated CD4+CD25h T cells could be expanded with IL-2 supplementation alone and maintained FOXP3 expression and suppressor function through the expansion. Our findings demonstrate that DNA demethylation can enhance the induction of human Treg cells and promise to solve one of the challenges with using Treg cells in therapeutic approaches.

CD4 down regulation and raft dissociation by the non-depleting YTS177 antibody hinder murine T helper cell activities.

Non-depleting YTS177 anti-CD4 monoclonal antibody (MoAb) has been reported to lead to antigen-specific immunotolerance in allograft transplantation and autoimmune diabetes, as well as possibly to inhibition of allergic inflammation in mice. However, the molecular mechanisms underlying hyporesponsive T cell responses induced by YTS177 MoAb remain elusive. Herein, we demonstrate that the YTS177 MoAb increases the levels of anergy factors p27(kip1) and Cbl-b, inhibits IL-2 production, and impairs calcium mobilization in activated T cells in vitro. YTS177 MoAb suppresses OVA-driven proliferation of DO11.10 CD4(+) T cells in vivo as well. Mechanistically, YTS177 MoAb induces tolerance by causing CD4 down-regulation through clathrin-dependent and raft dissociation. The results obtained in this study lead us to propose novel protective or curative approaches to CD4 T cell-mediated diseases.

Design of a heart rate controller for treadmill exercise using a recurrent fuzzy neural network.

BACKGROUND AND OBJECTIVE: In this study, we developed a computer controlled treadmill system using a recurrent fuzzy neural network heart rate controller (RFNNHRC). Treadmill speeds and inclines were controlled by corresponding control servo motors. The RFNNHRC was used to generate the control signals to automatically control treadmill speed and incline to minimize the user heart rate deviations from a preset profile. METHODS: The RFNNHRC combines a fuzzy reasoning capability to accommodate uncertain information and an artificial recurrent neural network learning process that corrects for treadmill system nonlinearities and uncertainties. Treadmill speeds and inclines are controlled by the RFNNHRC to achieve minimal heart rate deviation from a pre-set profile using adjustable parameters and an on-line learning algorithm that provides robust performance against parameter variations. The on-line learning algorithm of RFNNHRC was developed and implemented using a dsPIC 30F4011 DSP. RESULTS: Application of the proposed control scheme to heart rate responses of runners resulted in smaller fluctuations than those produced by using proportional integra control, and treadmill speeds and inclines were smoother. The present experiments demonstrate improved heart rate tracking performance with the proposed control scheme. CONCLUSIONS: The RFNNHRC scheme with adjustable parameters and an on-line learning algorithm was applied to a computer controlled treadmill system with heart rate control during treadmill exercise. Novel RFNNHRC structure and controller stability analyses were introduced. The RFNNHRC were tuned using a Lyapunov function to ensure system stability. The superior heart rate control with the proposed RFNNHRC scheme was demonstrated with various pre-set heart rates.

Expansion of regulatory T cells from umbilical cord blood and adult peripheral blood CD4(+)CD25 (+) T cells.

CD4(+)CD25(+) regulatory T cells (Treg), if properly expanded from umbilical cord blood (UCB), may provide a promising immunotherapeutic tool. Our previous data demonstrated that UCB CD4(+)CD25(+) T cells with 4-day stimulation have comparable phenotypes and suppressive function to that of adult peripheral blood (APB) CD4(+)CD25(+) T cells. We further examined whether 2-week culture would achieve higher expansion levels of Tregs. UCB CD4(+)CD25(+) T cells and their APB counterparts were stimulated with anti-CD3/anti-CD28 in the presence of IL-2 or IL-15 for 2 weeks. The cell proliferation and forkhead box P3 (FoxP3) expression were examined. The function of the expanded cells was then investigated by suppressive assay. IL-21 was applied to study whether it counteracts the function of UCB and APB CD4(+)CD25(+) T cells. The results indicate that UCB CD4(+)CD25(+) T cells expanded much better than their APB counterparts. IL-2 was superior to expand UCB and APB Tregs for 2 weeks than IL-15. FoxP3 expression which peaked on Day 10-14 was comparable. Most importantly, expanded UCB Tregs showed greater suppressive function in allogeneic mixed lymphocyte reaction. The addition of IL-21, however, counteracted the suppressive function of expanded UCB and APB Tregs. The results support using UCB as a source of Treg cells.

Suppression of B3GNT7 gene expression in colon adenocarcinoma and its potential effect in the metastasis of colon cancer cells.

The cell surface sialyl Lewis a (sLe(a)) and sialyl Lewis x (sLe(x)) antigens, which are built on the terminals of glyco-structures called poly-N-acetyllactosamine (LacNAc) chains, have been shown to play a critical role in the metastasis of colon cancer. In the present investigation, expression of the B3GNT7 gene, which encodes a ß-1,3-N-acetylglucosaminyltransferase that mainly acts on and extends sulfated poly-LacNAc chains, was found to be markedly suppressed during the oncogenetic processes associated with colon cancer. DNA methylation in the promoter region of the B3GNT7 gene was found to play a significant role in the suppression of the B3GNT7 gene in colon cancer cells. The results obtained from Transwell experiments and the nude mice xenograft model demonstrated that ectopic expression of the B3GNT7 gene in colon cancer cells diminished the migration capability and the liver-metastasis potential, respectively, of colon cancer cells. Flow cytometric analysis showed that expression of cell surface sLe(a) and sLe(x) antigens was decreased in colon cancer cells when the B3GNT7 gene was ectopically expressed. Taken together, the results of the present investigation suggest a link between suppression of B3GNT7 gene expression and elevation of sLe(a)/sLe(x) antigen expressions on the surface of cells and that this consequently promotes the metastasis potential of cancer cells as part of the colon cancer oncogenetic process.