Cytokine receptor clustering in sensory neurons with an engineered cytokine fusion protein triggers unique pain resolution pathways.

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.

11ß-HSD1 determines the extent of muscle atrophy in a model of acute exacerbation of COPD.

Muscle atrophy is an extrapulmonary complication of acute exacerbations (AE) in chronic obstructive pulmonary disease (COPD). The endogenous production and therapeutic application of glucocorticoids (GCs) have been implicated as drivers of muscle loss in AE-COPD. The enzyme 11 ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) activates GCs and contributes toward GC-induced muscle wasting. To explore the potential of 11ßHSD1 inhibition to prevent muscle wasting here, the objective of this study was to ascertain the contribution of endogenous GC activation and amplification by 11ßHSD1 in skeletal muscle wasting during AE-COPD. Emphysema was induced by intratracheal (IT) instillation of elastase to model COPD in WT and 11ßHSD1/KO mice, followed by vehicle or IT-LPS administration to mimic AE. µCT scans were obtained prior and at study endpoint 48 h following IT-LPS, to assess emphysema development and muscle mass changes, respectively. Plasma cytokine and GC profiles were determined by ELISA. In vitro, myonuclear accretion and cellular response to plasma and GCs were determined in C2C12 and human primary myotubes. Muscle wasting was exacerbated in LPS-11ßHSD1/KO animals compared with WT controls. RT-qPCR and western blot analysis showed elevated catabolic and suppressed anabolic pathways in muscle of LPS-11ßHSD1/KO animals relative to WTs. Plasma corticosterone levels were higher in LPS-11ßHSD1/KO animals, whereas C2C12 myotubes treated with LPS-11ßHSD1/KO plasma or exogenous GCs displayed reduced myonuclear accretion relative to WT counterparts. This study reveals that 11ß-HSD1 inhibition aggravates muscle wasting in a model of AE-COPD, suggesting that therapeutic inhibition of 11ß-HSD1 may not be appropriate to prevent muscle wasting in this setting.

Evaluation of the Sensitivity of Metabolic Profiling by Rapid Evaporative Ionization Mass Spectrometry: Toward More Radical Oral Cavity Cancer Resections.

Radical resection for patients with oral cavity cancer remains challenging. Rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical vapors has been reported for real-time classification of normal and tumor tissues for numerous surgical applications. However, the infiltrative pattern of invasion of oral squamous cell carcinomas (OSCC) challenges the ability of REIMS to detect low amounts of tumor cells. We evaluate REIMS sensitivity to determine the minimal amount of detected tumors cells during oral cavity cancer surgery. A total of 11 OSCC patients were included in this study. The tissue classification based on 185 REIMS ex vivo metabolic profiles from five patients was compared to histopathology classification using multivariate analysis and leave-one-patient-out cross-validation. Vapors were analyzed in vivo by REIMS during four glossectomies. Complementary desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was employed to map tissue heterogeneity on six oral cavity sections to support REIMS findings. REIMS sensitivity was assessed with a new cell-based assay consisting of mixtures of cell lines (tumor, myoblasts, keratinocytes). Our results depict REIMS classified tumor and soft tissues with 96.8% accuracy. In vivo REIMS generated intense mass spectrometric signals. REIMS detected 10% of tumor cells mixed with 90% myoblasts with 83% sensitivity and 82% specificity. DESI-MSI underlined distinct metabolic profiles of nerve features and a metabolic shift phosphatidylethanolamine PE(O-16:1/18:2))/cholesterol sulfate common to both mucosal maturation and OSCC differentiation. In conclusion, the assessment of tissue heterogeneity with DESI-MSI and REIMS sensitivity with cell mixtures characterized sensitive metabolic profiles toward in vivo tissue recognition during oral cavity cancer surgeries.

Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells.

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed-infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T-cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi-maturation of human monocytes-derived DCs (moDCs), and elevated levels of IL-10, IL-27 and IL-6 but not IL-12p70 and TNF-α. Consistently, HMOS-conditioned human moDCs promoted Treg generation from naïve CD4+ T cells. Interestingly, HMOS limited LPS-induced maturation of human moDCs, while maintained IL-10 and IL-27 secretion and reduced LPS-induced production of IL-12p70, IL-6 and TNF-α. Furthermore, HMOS+LPS-stimulated DCs induced a higher frequency of Tregs and increased IL-10 production, while a reduction in Tbet+Th1 frequency and IFN-γ production was detected as compared to LPS-DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC-SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.

Regulation of muscle atrophy by microRNAs: 'AtromiRs' as potential target in cachexia.

PURPOSE OF REVIEW: To provide an overview and describe the mode of action of miRNAs recently implicated in muscle atrophy, and discuss the challenges to explore their potential as putative therapeutic targets in cachexia. RECENT FINDINGS: Recent work showed differentially expressed miRNAs in skeletal muscle of patients with cachexia-associated diseases. Studies using experimental models revealed miRNA regulation of the anabolic IGF-1 and catabolic TGF- ß/myostatin pathways, and downstream protein synthesis and proteolysis signaling in control of muscle mass. SUMMARY: Cachexia is a complex metabolic condition associated with progressive body weight loss, wasting of skeletal muscle mass and decrease in muscle strength. MiRNAs play a central role in post-transcriptional gene regulation by targeting mRNAs, thereby coordinating and fine-tuning many cellular processes. MiRNA expression profiling studies of muscle biopsies have revealed differentially expressed miRNAs in patients with low muscle mass or cachexia. Evaluation in experimental models has revealed muscle atrophy, inhibition of protein synthesis and activation of proteolysis in response to modulation of specific miRNAs, termed 'atromiRs' in this review. These exciting findings call for further studies aimed at exploring the conservation of differentially expressed miRNAs across diseases accompanied by cachexia, identification of miRNA clusters and targets involved in muscle atrophy, and probing whether these miRNAs might be potential therapeutic targets for cachexia.

The effect of dietary components on inflammatory lung diseases - a literature review.

Anti-inflammatory treatment in chronic inflammatory lung diseases usually involves glucocorticosteroids. With patients suffering from serious side effects or becoming resistant, specific nutrients, that are suggested to positively influence disease progression, can be considered as new treatment options. The dietary inflammatory index is used to calculate effects of dietary components on inflammation and lung function to identify most potent dietary components, based on 162 articles. The positive effects of n-3 PUFAs and vitamin E on lung function can at least partially be explained by their anti-inflammatory effect. Many other dietary components showed only small or no effects on inflammation and/or lung function, although the number of weighted studies was often too small for a reliable assessment. Optimal beneficial dietary elements might reduce the required amounts of anti-inflammatory treatments, thereby decreasing both side effects and development of resistance as to improve quality of life of patients suffering from chronic inflammatory lung diseases.

Cachexia and Sarcopenia in Oligometastatic Non-Small Cell Lung Cancer: Making a Potential Curable Disease Incurable?

Among patients with advanced NSCLC, there is a group of patients with synchronous oligometastatic disease (sOMD), defined as a limited number of metastases detected at the time of diagnosis. As cachexia and sarcopenia are linked to poor survival, incorporating this information could assist clinicians in determining whether a radical treatment should be administered. In a retrospective multicenter study, including all patients with adequately staged (FDG-PET, brain imaging) sOMD according to the EORTC definition, we aimed to assess the relationship between cachexia and/or sarcopenia and survival. Of the 439 patients that were identified between 2015 and 2021, 234 met the criteria for inclusion and were included. The median age of the cohort was 67, 52.6% were male, and the median number of metastasis was 1. Forty-six (19.7%) patients had cachexia, thirty-four (14.5%) had sarcopenia and twenty-one (9.0%) had both. With a median follow-up of 49.7 months, median PFS and OS were 8.6 and 17.3 months, respectively. Moreover, a trend toward longer PFS was found in patients without cachexia and sarcopenia compared to those with cachexia and/or sarcopenia. In multivariate analysis, cachexia and sarcopenia were not associated with an inferior survival, irrespective of receiving radical treatment. High CRP was associated with inferior survival and could be a prognostic factor, helping the decision of clinicians in selecting patients who may benefit from the addition of LRT. However, despite the homogeneous definition of oligometastatic disease and the adequate staging, our subgroups were small. Therefore, further studies are needed to better understand our hypothesis and generating findings.

Targeted nutritional intervention attenuates experimental lung cancer cachexia.

BACKGROUND: Cachexia, a syndrome with high prevalence in non-small cell lung cancer patients, impairs quality of life and reduces tolerance and responsiveness to cancer therapy resulting in decreased survival. Optimal nutritional care is pivotal in the treatment of cachexia and a recommended cornerstone of multimodal therapy. Here, we investigated the therapeutic effect of an intervention diet consisting of a specific combination of high protein, leucine, fish oil, vitamin D, galacto-oligosaccharides, and fructo-oligosaccharides on the development and progression of cachexia in an orthotopic lung cancer mouse model. METHODS: Eleven-week-old male 129S2/Sv mice were orthotopically implanted with 344P lung epithelial tumour cells or vehicle (control). Seven days post-implantation tumour-bearing (TB) mice were allocated to either intervention- or isocaloric control diet. Cachexia was defined as 5 days of consecutive body weight loss, after which mice were euthanized for tissue analyses. RESULTS: TB mice developed cachexia accompanied by significant loss of skeletal muscle mass and epididymal fat mass compared with sham operated mice. The cachectic endpoint was significantly delayed (46.0 ± 15.2 vs. 34.7 ± 11.4 days), and the amount (-1.57 ± 0.62 vs. -2.13 ± 0.57 g) and progression (-0.26 ± 0.14 vs. -0.39 ± 0.11 g/day) of body weight loss were significantly reduced by the intervention compared with control diet. Moreover, systemic inflammation (pentraxin-2 plasma levels) and alterations in molecular markers for proteolysis and protein synthesis, indicative of muscle atrophy signalling in TB-mice, were suppressed in skeletal muscle by the intervention diet. CONCLUSIONS: Together, these data demonstrate the potential of this multinutrient intervention, targeting multiple components of cachexia, as integral part of lung cancer management.

A novel orthotopic mouse model replicates human lung cancer cachexia.

INTRODUCTION: Cancer cachexia, highly prevalent in lung cancer, is a debilitating syndrome characterized by involuntary loss of skeletal muscle mass and is associated with poor clinical outcome, decreased survival and negative impact on tumour therapy. Various lung tumour-bearing animal models have been used to explore underlying mechanisms of cancer cachexia. However, these models do not simulate anatomical and immunological features key to lung cancer and associated muscle wasting. Overcoming these shortcomings is essential to translate experimental findings into the clinic. We therefore evaluated whether a syngeneic, orthotopic lung cancer mouse model replicates systemic and muscle-specific alterations associated with human lung cancer cachexia. METHODS: Immune competent, 11 weeks old male 129S2/Sv mice, were randomly allocated to either (1) sham control group or (2) tumour-bearing group. Syngeneic lung epithelium-derived adenocarcinoma cells (K-rasG12D ; p53R172HΔG ) were inoculated intrapulmonary into the left lung lobe of the mice. Body weight and food intake were measured daily. At baseline and weekly after surgery, grip strength was measured and tumour growth and muscle volume were assessed using micro cone beam CT imaging. After reaching predefined surrogate survival endpoint, animals were euthanized, and skeletal muscles of the lower hind limbs were collected for biochemical analysis. RESULTS: Two-third of the tumour-bearing mice developed cachexia based on predefined criteria. Final body weight (-13.7 ± 5.7%; P < 0.01), muscle mass (-13.8 ± 8.1%; P < 0.01) and muscle strength (-25.5 ± 10.5%; P < 0.001) were reduced in cachectic mice compared with sham controls and median survival time post-surgery was 33.5 days until humane endpoint. Markers for proteolysis, both ubiquitin proteasome system (Fbxo32 and Trim63) and autophagy-lysosomal pathway (Gabarapl1 and Bnip3), were significantly upregulated, whereas markers for protein synthesis (relative phosphorylation of Akt, S6 and 4E-BP1) were significantly decreased in the skeletal muscle of cachectic mice compared with control. The cachectic mice exhibited increased pentraxin-2 (P < 0.001) and CXCL1/KC (P < 0.01) expression levels in blood plasma and increased mRNA expression of IκBα (P < 0.05) in skeletal muscle, indicative for the presence of systemic inflammation. Strikingly, RNA sequencing, pathway enrichment and miRNA expression analyses of mouse skeletal muscle strongly mirrored alterations observed in muscle biopsies of patients with lung cancer cachexia. CONCLUSIONS: We developed an orthotopic model of lung cancer cachexia in immune competent mice. Because this model simulates key aspects specific to cachexia in lung cancer patients, it is highly suitable to further investigate the underlying mechanisms of lung cancer cachexia and to test the efficacy of novel intervention strategies.

Physical exercise at the crossroad between muscle wasting and the immune system: implications for lung cancer cachexia.

Cachexia is a syndrome characterized by involuntary weight loss and wasting of skeletal muscle mass. It is associated with worse overall survival and quality of life. The cancer-induced systemic inflammation and the consequent host derived catabolic stimuli, trigger cachexia by inhibiting muscle protein synthesis and enhancing muscle catabolism. The muscle itself may further promote chronic inflammation, introducing a vicious catabolic circle. Nutritional support alone plays a limited role in the treatment of cancer cachexia and should be combined with other interventions. Physical exercise lowers systemic inflammation and promotes muscle anabolism. It also attenuates the age-related physical decline in elderly and it might counteract the muscle wasting induced by the cancer cachexia syndrome. This review describes how cancer-induced systemic inflammation promotes muscle wasting and whether physical exercise may represent a suitable treatment for cancer-induced cachexia, particularly in patients with non-small cell lung cancer. We summarized pre-clinical and clinical studies investigating whether physical exercise would improve muscle performance and whether this improvement would translate in a clinically meaningful benefit for patients with cancer, in terms of survival and quality of life. Moreover, this review describes the results of studies investigating the interplay between physical exercise and the immune system, including the role of the intestinal microbiota.

Ovarian cancer ascites induces skeletal muscle wasting in vitro and reflects sarcopenia in patients.

BACKGROUND: Cachexia-associated skeletal muscle wasting or 'sarcopenia' is highly prevalent in ovarian cancer and contributes to poor outcome. Drivers of cachexia-associated sarcopenia in ovarian cancer remain elusive, underscoring the need for novel and better models to identify tumour factors inducing sarcopenia. We aimed to assess whether factors present in ascites of sarcopenic vs. non-sarcopenic ovarian cancer patients differentially affect protein metabolism in skeletal muscle cells and to determine if these effects are correlated to cachexia-related patient characteristics. METHODS: Fifteen patients with an ovarian mass and ascites underwent extensive physical screening focusing on cachexia-related parameters. Based on computed tomography-based body composition imaging, six cancer patients were classified as sarcopenic and six were not; three patients with a benign condition served as an additional non-sarcopenic control group. Ascites was collected, and concentrations of cachexia-associated factors were assessed by enzyme-linked immunosorbent assay. Subsequently, ascites was used for in vitro exposure of C2C12 myotubes followed by measurements of protein synthesis and breakdown by radioactive isotope tracing, qPCR-based analysis of atrophy-related gene expression, and NF-κB activity reporter assays. RESULTS: C2C12 protein synthesis was lower after exposure to ascites from sarcopenic patients (sarcopenia 3.1 ± 0.1 nmol/h/mg protein vs. non-sarcopenia 5.5 ± 0.2 nmol/h/mg protein, P < 0.01), and protein breakdown rates tended to be higher (sarcopenia 31.2 ± 5.2% vs. non-sarcopenia 20.9 ± 1.9%, P = 0.08). Ascites did not affect MuRF1, Atrogin-1, or REDD1 expression of C2C12 myotubes, but NF-κB activity was specifically increased in cells exposed to ascites from sarcopenic patients (sarcopenia 2.2 ± 0.4-fold compared with control vs. non-sarcopenia 1.2 ± 0.2-fold compared with control, P = 0.01). Protein synthesis and breakdown correlated with NF-κB activity (rs  = -0.60, P = 0.03 and rs  = 0.67, P = 0.01, respectively). The skeletal muscle index of the ascites donors was also correlated to both in vitro protein synthesis (rs  = 0.70, P = 0.005) and protein breakdown rates (rs  = -0.57, P = 0.04). CONCLUSIONS: Ascites of sarcopenic ovarian cancer patients induces pronounced skeletal muscle protein metabolism changes in C2C12 cells that correlate with clinical muscle measures of the patient and that are characteristic of cachexia. The use of ascites offers a new experimental tool to study the impact of both tumour-derived and systemic factors in various cachexia model systems, enabling identification of novel drivers of tissue wasting in ovarian cancer.

Deep Learning Based Automated Orthotopic Lung Tumor Segmentation in Whole-Body Mouse CT-Scans.

Lung cancer is the leading cause of cancer related deaths worldwide. The development of orthotopic mouse models of lung cancer, which recapitulates the disease more realistically compared to the widely used subcutaneous tumor models, is expected to critically aid the development of novel therapies to battle lung cancer or related comorbidities such as cachexia. However, follow-up of tumor take, tumor growth and detection of therapeutic effects is difficult, time consuming and requires a vast number of animals in orthotopic models. Here, we describe a solution for the fully automatic segmentation and quantification of orthotopic lung tumor volume and mass in whole-body mouse computed tomography (CT) scans. The goal is to drastically enhance the efficiency of the research process by replacing time-consuming manual procedures with fast, automated ones. A deep learning algorithm was trained on 60 unique manually delineated lung tumors and evaluated by four-fold cross validation. Quantitative performance metrics demonstrated high accuracy and robustness of the deep learning algorithm for automated tumor volume analyses (mean dice similarity coefficient of 0.80), and superior processing time (69 times faster) compared to manual segmentation. Moreover, manual delineations of the tumor volume by three independent annotators was sensitive to bias in human interpretation while the algorithm was less vulnerable to bias. In addition, we showed that besides longitudinal quantification of tumor development, the deep learning algorithm can also be used in parallel with the previously published method for muscle mass quantification and to optimize the experimental design reducing the number of animals needed in preclinical studies. In conclusion, we implemented a method for fast and highly accurate tumor quantification with minimal operator involvement in data analysis. This deep learning algorithm provides a helpful tool for the noninvasive detection and analysis of tumor take, tumor growth and therapeutic effects in mouse orthotopic lung cancer models.

Nutritional Interventions in Cancer Cachexia: Evidence and Perspectives From Experimental Models.

Cancer cachexia is a complex metabolic syndrome characterized by involuntary skeletal muscle loss and is associated with poor clinical outcome, decreased survival and negatively influences cancer therapy. No curative treatments are available for cancer cachexia, but nutritional intervention is recommended as a cornerstone of multimodal therapy. Optimal nutritional care is pivotal in the treatment of cancer cachexia, and the effects of nutrients may extend beyond provision of adequate energy uptake, targeting different mechanisms or metabolic pathways that are affected or deregulated by cachexia. The evidence to support this notion derived from nutritional intervention studies in experimental models of cancer cachexia is systematically discussed in this review. Moreover, experimental variables and readout parameters to determine skeletal muscle wasting and cachexia are methodologically evaluated to allow critical comparison of similar studies. Single- and multinutrient intervention studies including qualitative modulation of dietary protein, dietary fat, and supplementation with specific nutrients, such as carnitine and creatine, were reviewed for their efficacy to counteract muscle mass loss and its underlying mechanisms in experimental cancer cachexia. Numerous studies showed favorable effects on impaired protein turnover and related metabolic abnormalities of nutritional supplementation in parallel with a beneficial impact on cancer-induced muscle wasting. The combination of high quality nutrients in a multitargeted, multinutrient approach appears specifically promising, preferentially as a multimodal intervention, although more studies investigating the optimal quantity and combination of nutrients are needed. During the review process, a wide variation in timing, duration, dosing, and route of supplementation, as well as a wide variation in animal models were observed. Better standardization in dietary design, and the development of experimental models that better recapitulate the etiology of human cachexia, will further facilitate successful translation of experimentally-based multinutrient, multimodal interventions into clinical practice.

Automated CT-derived skeletal muscle mass determination in lower hind limbs of mice using a 3D U-Net deep learning network.

The loss of skeletal muscle mass is recognized as a complication of several chronic diseases and is associated with increased mortality and a decreased quality of life. Relevant and reliable animal models in which muscle wasting can be monitored noninvasively over time are instrumental to investigate and develop new therapies. In this work, we developed a fully automatic deep learning algorithm for segmentation of micro cone beam computed tomography images of the lower limb muscle complex in mice and subsequent muscle mass calculation. A deep learning algorithm was trained on manually segmented data from 32 mice. Muscle wet mass measurements were obtained from 47 mice and served as a data set for model validation and reverse model validation. The automatic algorithm performance was ~150 times faster than manual segmentation. Reverse validation of the algorithm showed high quantitative metrics (i.e., a Dice similarity coefficient of 0.93, a Hausdorff distance of 0.4 mm, and a center of mass displacement of 0.1 mm), substantiating the robustness and accuracy of the model. A high correlation (R2 = 0.92) was obtained between the computed tomography-derived muscle mass measurements and the muscle wet masses. Longitudinal follow-up revealed time-dependent changes in muscle mass that separated control from lung tumor-bearing mice, which was confirmed as cachexia. In conclusion, this deep learning model for automated assessment of the lower limb muscle complex provides highly accurate noninvasive longitudinal evaluation of skeletal muscle mass. Furthermore, it facilitates the workflow and increases the amount of data derived from mouse studies while reducing the animal numbers.NEW & NOTEWORTHY This deep learning application enables highly accurate noninvasive longitudinal evaluation of skeletal muscle mass changes in mice with minimal requirement for operator involvement in the data analysis. It provides a unique opportunity to increase and analyze the amount of data derived from animal studies automatically while reducing animal numbers and analytical workload.

Identification of microRNAs in skeletal muscle associated with lung cancer cachexia.

BACKGROUND: Cachexia, highly prevalent in patients with non-small cell lung cancer (NSCLC), impairs quality of life and is associated with reduced tolerance and responsiveness to cancer therapy and decreased survival. MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in post-transcriptional gene regulation. Changes in intramuscular levels of miRNAs have been implicated in muscle wasting conditions. Here, we aimed to identify miRNAs that are differentially expressed in skeletal muscle of cachectic lung cancer patients to increase our understanding of cachexia and to allow us to probe their potential as therapeutic targets. METHODS: A total of 754 unique miRNAs were profiled and analysed in vastus lateralis muscle biopsies of newly diagnosed treatment-naïve NSCLC patients with cachexia (n = 8) and age-matched and sex-matched healthy controls (n = 8). miRNA expression analysis was performed using a TaqMan MicroRNA Array. In silico network analysis was performed on all significant differentially expressed miRNAs. Differential expression of the top-ranked miRNAs was confirmed using reverse transcription-quantitative real-time PCR in an extended group (n = 48) consisting of NSCLC patients with (n = 15) and without cachexia (n = 11) and healthy controls (n = 22). Finally, these miRNAs were subjected to univariate and multivariate Cox proportional hazard analysis using overall survival and treatment-induced toxicity data obtained during the follow-up of this group of patients. RESULTS: We identified 28 significant differentially expressed miRNAs, of which five miRNAs were up-regulated and 23 were down-regulated. In silico miRNA-target prediction analysis showed 158 functional gene targets, and pathway analysis identified 22 pathways related to the degenerative or regenerative processes of muscle tissue. Subsequently, the expression of six top-ranked miRNAs was measured in muscle biopsies of the entire patient group. Five miRNAs were detectable with reverse transcription-quantitative real-time PCR analysis, and their altered expression (expressed as fold change, FC) was confirmed in muscle of cachectic NSCLC patients compared with healthy control subjects: miR-424-5p (FC = 4.5), miR-424-3p (FC = 12), miR-450a-5p (FC = 8.6), miR-144-5p (FC = 0.59), and miR-451a (FC = 0.57). In non-cachectic NSCLC patients, only miR-424-3p was significantly increased (FC = 5.6) compared with control. Although the statistical support was not sufficient to imply these miRNAs as individual predictors of overall survival or treatment-induced toxicity, when combined in multivariate analysis, miR-450-5p and miR-451a resulted in a significant stratification between short-term and long-term survival. CONCLUSIONS: We identified differentially expressed miRNAs putatively involved in lung cancer cachexia. These findings call for further studies to investigate the causality of these miRNAs in muscle atrophy and the mechanisms underlying their differential expression in lung cancer cachexia.

Early-Life Nutritional Factors and Mucosal Immunity in the Development of Autoimmune Diabetes.

Type 1 diabetes (T1D) is an immune-mediated disease with a strong genetic basis but might be influenced by non-genetic factors such as microbiome development that "programs" the immune system during early life as well. Factors influencing pathogenesis, including a leaky intestinal mucosal barrier, an aberrant gut microbiota composition, and altered immune responsiveness, offer potential targets for prevention and/or treatment of T1D through nutritional or pharmacologic means. In this review, nutritional approaches during early life in order to protect against T1D development have been discussed. The critical role of tolerogenic dendritic cells in central and peripheral tolerance has been emphasized. In addition, since the gut microbiota affects the development of T1D through short-chain fatty acid (SCFA)-dependent mechanisms, we hypothesize that nutritional intervention boosting SCFA production may be used as a novel prevention strategy. Current retrospective evidence has suggested that exclusive and prolonged breastfeeding might play a protective role against the development of T1D. The beneficial properties of human milk are possibly attributed to its bioactive components such as unique immune-modulatory components human milk oligosaccharides and metabolites derived thereof, including SCFAs. These components might play a key role in healthy immune development and creating a fit and resilient immune system in early and later life.