TRPV4-Expressing Tissue-Resident Macrophages Regulate the Function of Collecting Lymphatic Vessels via Thromboxane A2 Receptors in Lymphatic Muscle Cells.

Rationale: TRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and tissue fibrosis. These are key features in the pathophysiology of lymphatic system diseases, including lymphedema and lipedema; however, the role of TRPV4 channels in the lymphatic system remains largely unexplored. TRPV4 channels are calcium permeable, non-selective cation channels that are activated by diverse stimuli, including shear stress, stretch, temperature, and cell metabolites, which may regulate lymphatic contractile function. Objective: To characterize the expression of TRPV4 channels in collecting lymphatic vessels and to determine the extent to which these channels regulate the contractile function of lymphatics. Methods and Results: Pressure myography on intact, isolated, and cannulated lymphatic vessels showed that pharmacological activation of TRPV4 channels with GSK1016790A (GSK101) led to contractile dysregulation. The response to GSK101 was multiphasic and included, 1) initial robust constriction that was sustained for ≥1 minute and in some instances remained for ≥4 minutes; and 2) subsequent vasodilation and partial or complete inhibition of lymphatic contractions associated with release of nitric oxide. The functional response to activation of TRPV4 channels displayed differences across lymphatics from four anatomical regions, but these differences were consistent across different species (mouse, rat, and non-human primate). Importantly, similar responses were observed following activation of TRPV4 channels in arterioles. The initial and sustained constriction was prevented with the COX inhibitor, indomethacin. We generated a controlled and spatially defined single-cell RNA sequencing (scRNAseq) dataset from intact and microdissected collecting lymphatic vessels. Our data uncovered a subset of macrophages displaying the highest expression of Trpv4 compared to other cell types within and surrounding the lymphatic vessel wall. These macrophages displayed a transcriptomic profile consistent with that of tissue-resident macrophages (TRMs), including differential expression of Lyve1 , Cd163 , Folr2 , Mrc1 , Ccl8 , Apoe , Cd209f , Cd209d , and Cd209g ; and at least half of these macrophages also expressed Timd4. This subset of macrophages also highly expressed Txa2s , which encodes the thromboxane A2 (TXA2) synthase. Inhibition of TXA2 receptors (TXA2Rs) prevented TRPV4-mediated contractile dysregulation. TXA2R activation on LMCs caused an increase in mobilization of calcium from intracellular stores through Ip3 receptors which promoted store operated calcium entry and vasoconstriction. Conclusions: Clinical studies have linked cancer-related lymphedema with an increased infiltration of macrophages. While these macrophages have known anti-inflammatory and pro-lymphangiogenic roles, as well as promote tissue repair, our results point to detrimental effects to the pumping capacity of collecting lymphatic vessels mediated by activation of TRPV4 channels in macrophages. Pharmacological targeting of TRPV4 channels in LYVE1-expressing macrophages or pharmacological targeting of TXA2Rs may offer novel therapeutic strategies to improve lymphatic pumping function and lymph transport in lymphedema.

Breast cancer-related lymphedema results in impaired epidermal differentiation and tight junction dysfunction.

Breast cancer-related lymphedema (BCRL) is characterized by skin changes, swelling, fibrosis, and recurrent skin infections. Clinical studies have suggested that lymphedema results in skin barrier defects; however, the underlying cellular mechanisms and the effects of bacterial contamination on skin barrier function remain unknown. In matched biopsies from patients with unilateral BCRL, we observed decreased expression of filaggrin and the tight junction protein zona occludens-1 (ZO-1) in skin affected by moderate lymphedema, or by subclinical lymphedema in which dermal backflow of lymph was identified by indocyanine green lymphography, relative to controls (areas without backflow and from the unaffected arm). In vitro stimulation of keratinocytes with lymph fluid obtained from patients undergoing lymphedema surgery led to the same changes, as well as increased expression of keratin 14, a marker of immature keratinocytes. Finally, using mouse models of lymphedema, we showed that like the clinical scenario, the expression of skin barrier proteins was decreased relative to normal skin and that colonization with S. epidermidis bacteria amplified this effect, as well as lymphedema severity. Taken together, our findings suggest that lymphatic fluid stasis contributes to skin barrier dysfunction in lymphedema.

TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Lymphatic-Dependent Modulation of the Sensitization and Elicitation Phases of Contact Hypersensitivity.

Allergic contact dermatitis is a common inflammatory skin disease comprising 2 phases. During sensitization, immune cells are activated by exposure to various allergens, whereas repeated antigen exposure induces local inflammation during elicitation. In this study, we utilized mouse models lacking lymphatics in different skin regions to characterize the role of lymphatics separately in the 2 phases, using contact hypersensitivity as a model of human allergic inflammatory skin diseases. Lymphatic-deficient mice exhibited no major difference to single antigen exposure compared to controls. However, mice lacking lymphatics in both phases displayed reduced inflammation after repeated antigen exposure. Similarly, diminished immune response was observed in mice lacking lymphatics only in sensitization, whereas the absence of lymphatics only in the elicitation phase resulted in a more pronounced inflammatory immune response. This exaggerated inflammation is driven by neutrophils impacting regulatory T cell number. Collectively, our results demonstrate that skin lymphatics play an important but distinct role in the 2 phases of contact hypersensitivity. During sensitization, lymphatics contribute to the development of the antigen-specific immunization, whereas in elicitation, they moderate the inflammatory response and leukocyte infiltration in a neutrophil-dependent manner. These findings underscore the need for novel therapeutic strategies targeting the lymphatics in the context of allergic skin diseases.

Therapy-Induced Senescence Contributes to the Efficacy of Abemaciclib in Patients with Dedifferentiated Liposarcoma.

PURPOSE: We conducted research on CDK4/6 inhibitors (CDK4/6i) simultaneously in the preclinical and clinical spaces to gain a deeper understanding of how senescence influences tumor growth in humans. PATIENTS AND METHODS: We coordinated a first-in-kind phase II clinical trial of the CDK4/6i abemaciclib for patients with progressive dedifferentiated liposarcoma (DDLS) with cellular studies interrogating the molecular basis of geroconversion. RESULTS: Thirty patients with progressing DDLS enrolled and were treated with 200 mg of abemaciclib twice daily. The median progression-free survival was 33 weeks at the time of the data lock, with 23 of 30 progression-free at 12 weeks (76.7%, two-sided 95% CI, 57.7%-90.1%). No new safety signals were identified. Concurrent preclinical work in liposarcoma cell lines identified ANGPTL4 as a necessary late regulator of geroconversion, the pathway from reversible cell-cycle exit to a stably arrested inflammation-provoking senescent cell. Using this insight, we were able to identify patients in which abemaciclib induced tumor cell senescence. Senescence correlated with increased leukocyte infiltration, primarily CD4-positive cells, within a month of therapy. However, those individuals with both senescence and increased TILs were also more likely to acquire resistance later in therapy. These suggest that combining senolytics with abemaciclib in a subset of patients may improve the duration of response. CONCLUSIONS: Abemaciclib was well tolerated and showed promising activity in DDLS. The discovery of ANGPTL4 as a late regulator of geroconversion helped to define how CDK4/6i-induced cellular senescence modulates the immune tumor microenvironment and contributes to both positive and negative clinical outcomes. See related commentary by Weiss et al., p. 649.

Placental microRNA methylome signatures may serve as biomarkers and therapeutic targets for prenatally opioid-exposed infants with neonatal opioid withdrawal syndrome.

Introduction: The neonate exposed to opioids in utero faces a constellation of withdrawal symptoms postpartum commonly called neonatal opioid withdrawal syndrome (NOWS). The incidence of NOWS has increased in recent years due to the opioid epidemic. MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. Epigenetic variations in microRNAs (miRNAs) and their impact on addiction-related processes is a rapidly evolving area of research. Methods: The Illumina Infinium Methylation EPIC BeadChip was used to analyze DNA methylation levels of miRNA-encoding genes in 96 human placental tissues to identify miRNA gene methylation profiles as-sociated with NOWS: 32 from mothers whose prenatally opioid-exposed infants required pharmacologic management for NOWS, 32 from mothers whose prenatally opioid-exposed infants did not require treat-ment for NOWS, and 32 unexposed controls. Results: The study identified 46 significantly differentially methylated (FDR p-value ≤ 0.05) CpGs associated with 47 unique miRNAs, with a receiver operating characteristic (ROC) area under the curve (AUC) ≥0.75 including 28 hypomethylated and 18 hypermethylated CpGs as potentially associated with NOWS. These dysregulated microRNA methylation patterns may be a contributing factor to NOWS pathogenesis. Conclusion: This is the first study to analyze miRNA methylation profiles in NOWS infants and illustrates the unique role miRNAs might have in diagnosing and treating the disease. Furthermore, these data may provide a step toward feasible precision medicine for NOWS babies as well.

The Future of Lymphedema: Potential Therapeutic Targets for Treatment.

Purpose of Review: This review aims to summarize the current knowledge regarding the pharmacological interventions studied in both experimental and clinical trials for secondary lymphedema. Recent Findings: Lymphedema is a progressive disease that results in tissue swelling, pain, and functional disability. The most common cause of secondary lymphedema in developed countries is an iatrogenic injury to the lymphatic system during cancer treatment. Despite its high incidence and severe sequelae, lymphedema is usually treated with palliative options such as compression and physical therapy. However, recent studies on the pathophysiology of lymphedema have explored pharmacological treatments in preclinical and early phase clinical trials. Summary: Many potential treatment options for lymphedema have been explored throughout the past two decades including systemic agents and topical approaches to decrease the potential toxicity of systemic treatment. Treatment strategies including lymphangiogenic factors, anti-inflammatory agents, and anti-fibrotic therapies may be used independently or in conjunction with surgical approaches.

Skin microbiome alterations in upper extremity secondary lymphedema.

Lymphedema is a chronic condition that commonly occur from lymphatic injury following surgical resection of solid malignancies. While many studies have centered on the molecular and immune pathways that perpetuate lymphatic dysfunction, the role of the skin microbiome in lymphedema development remains unclear. In this study, skin swabs collected from normal and lymphedema forearms of 30 patients with unilateral upper extremity lymphedema were analyzed by 16S ribosomal RNA sequencing. Statistical models for microbiome data were utilized to correlate clinical variables with microbial profiles. Overall, 872 bacterial taxa were identified. There were no significant differences in microbial alpha diversity of the colonizing bacteria between normal and lymphedema skin samples (p = 0.25). Notably, for patients without a history of infection, a one-fold change in relative limb volume was significantly associated with a 0.58-unit increase in Bray-Curtis microbial distance between paired limbs (95%CI = 0.11,1.05, p = 0.02). Additionally, several genera, including Propionibacterium and Streptococcus, demonstrated high variability between paired samples. In summary, we demonstrate high compositional heterogeneity in the skin microbiome in upper extremity secondary lymphedema, supporting future studies into the role of host-microbe interactions on lymphedema pathophysiology.

Topical captopril: a promising treatment for secondary lymphedema.

Transforming growth factor-beta 1 (TGF-ß1)-mediated tissue fibrosis is an important regulator of lymphatic dysfunction in secondary lymphedema. However, TGF-ß1 targeting can cause toxicity and autoimmune complications, limiting clinical utility. Angiotensin II (Ang II) modulates intracellular TGF-ß1 signaling, and inhibition of Ang II production using angiotensin-converting enzyme (ACE) inhibitors, such as captopril, has antifibrotic efficacy in some pathological settings. Therefore, we analyzed the expression of ACE and Ang II in clinical lymphedema biopsy specimens from patients with unilateral breast cancer-related lymphedema (BCRL) and mouse models, and found that cutaneous ACE expression is increased in lymphedematous tissues. Furthermore, topical captopril decreases fibrosis, activation of intracellular TGF-ß1 signaling pathways, inflammation, and swelling in mouse models of lymphedema. Captopril treatment also improves lymphatic function and immune cell trafficking by increasing collecting lymphatic pumping. Our results show that the renin-angiotensin system in the skin plays an important role in the regulation of fibrosis in lymphedema, and inhibition of this signaling pathway may hold merit for treating lymphedema.

Keratinocytes coordinate inflammatory responses and regulate development of secondary lymphedema.

Epidermal changes are histological hallmarks of secondary lymphedema, but it is unknown if keratinocytes contribute to its pathophysiology. Using clinical lymphedema specimens and mouse models, we show that keratinocytes play a primary role in lymphedema development by producing T-helper 2 (Th2) -inducing cytokines. Specifically, we find that keratinocyte proliferation and expression of protease-activated receptor 2 (PAR2) are early responses following lymphatic injury and regulate the expression of Th2-inducing cytokines, migration of Langerhans cells, and skin infiltration of Th2-differentiated T cells. Furthermore, inhibition of PAR2 activation with a small molecule inhibitor or the proliferation inhibitor teriflunomide (TF) prevents activation of keratinocytes stimulated with lymphedema fluid. Finally, topical TF is highly effective for decreasing swelling, fibrosis, and inflammation in a preclinical mouse model. Our findings suggest that lymphedema is a chronic inflammatory skin disease, and topically targeting keratinocyte activation may be a clinically effective therapy for this condition.

The Vicious Circle of Stasis, Inflammation, and Fibrosis in Lymphedema.

SUMMARY: Lymphedema is a progressive disease of the lymphatic system arising from impaired lymphatic drainage, accumulation of interstitial fluid, and fibroadipose deposition. Secondary lymphedema resulting from cancer treatment is the most common form of the disease in developed countries, affecting 15% to 40% of patients with breast cancer after lymph node dissection. Despite recent advances in microsurgery, outcomes remain variable and, in some cases, inadequate. Thus, development of novel treatment strategies is an important goal. Research over the past decade suggests that lymphatic injury initiates a chronic inflammatory response that regulates the pathophysiology of lymphedema. T-cell inflammation plays a key role in this response. In this review, the authors highlight the cellular and molecular mechanisms of lymphedema and discuss promising preclinical therapies.

Dysregulation of Lymphatic Endothelial VEGFR3 Signaling in Disease.

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), a receptor tyrosine kinase encoded by the FLT4 gene, plays a significant role in the morphogenesis and maintenance of lymphatic vessels. Under both normal and pathologic conditions, VEGF-C and VEGF-D bind VEGFR3 on the surface of lymphatic endothelial cells (LECs) and induce lymphatic proliferation, migration, and survival by activating intracellular PI3K-Akt and MAPK-ERK signaling pathways. Impaired lymphatic function and VEGFR3 signaling has been linked with a myriad of commonly encountered clinical conditions. This review provides a brief overview of intracellular VEGFR3 signaling in LECs and explores examples of dysregulated VEGFR3 signaling in various disease states, including (1) lymphedema, (2) tumor growth and metastasis, (3) obesity and metabolic syndrome, (4) organ transplant rejection, and (5) autoimmune disorders. A more complete understanding of the molecular mechanisms underlying the lymphatic pathology of each disease will allow for the development of novel strategies to treat these chronic and often debilitating illnesses.

Placental cytochrome P450 methylomes in infants exposed to prenatal opioids: exploring the effects of neonatal opioid withdrawal syndrome on health horizons.

Background: Neonatal opioid withdrawal syndrome (NOWS), arises due to increased opioid use during pregnancy. Cytochrome P450 (CYP) enzymes play a pivotal role in metabolizing a wide range of substances in the human body, including opioids, other drugs, toxins, and endogenous compounds. The association between CYP gene methylation and opioid effects is unexplored and it could offer promising insights. Objective: To investigate the impact of prenatal opioid exposure on disrupted CYPs in infants and their anticipated long-term clinical implications. Study Design: DNA methylation levels of CYP genes were analyzed in a cohort of 96 placental tissues using Illumina Infinium MethylationEPIC (850 k) BeadChips. This involved three groups of placental tissues: 32 from mothers with infants exposed to opioids prenatally requiring pharmacologic treatment for NOWS, 32 from mothers with prenatally opioid-exposed infants not needing NOWS treatment, and 32 from unexposed control mothers. Results: The study identified 20 significantly differentially methylated CpG sites associated with 17 distinct CYP genes, with 14 CpGs showing reduced methylation across 14 genes (CYP19A1, CYP1A2, CYP4V2, CYP1B1, CYP24A1, CYP26B1, CYP26C1, CYP2C18, CYP2C9, CYP2U1, CYP39A1, CYP2R1, CYP4Z1, CYP2D7P1 and), while 8 exhibited hypermethylation (CYP51A1, CYP26B1, CYP2R1, CYP2U1, CYP4X1, CYP1A2, CYP2W1, and CYP4V2). Genes such as CYP1A2, CYP26B1, CYP2R1, CYP2U1, and CYP4V2 exhibited both increased and decreased methylation. These genes are crucial for metabolizing eicosanoids, fatty acids, drugs, and diverse substances. Conclusion: The study identified profound methylation changes in multiple CYP genes in the placental tissues relevant to NOWS. This suggests that disruption of DNA methylation patterns in CYP transcripts might play a role in NOWS and may serve as valuable biomarkers, suggesting a future pathway for personalized treatment. Further research is needed to confirm these findings and explore their potential for diagnosis and treatment.

Tumor-Associated Lymphatics Upregulate MHC-II to Suppress Tumor-Infiltrating Lymphocytes.

Steady-state lymphatic endothelial cells (LECs) can induce peripheral tolerance by presenting endogenous antigens on MHC class I (MHC-I) molecules. Recent evidence suggests that lymph node LECs can cross-present tumor antigens on MHC-I to suppress tumor-specific CD8+ T cells. Whether LECs can act as immunosuppressive cells in an MHC-II dependent manner in the local tumor microenvironment (TME) is not well characterized. Using murine heterotopic and spontaneous tumor models, we show that LECs in the TME increase MHC-II expression in the context of increased co-inhibitory signals. We provide evidence that tumor lymphatics in human melanoma and breast cancer also upregulate MHC-II compared to normal tissue lymphatics. In transgenic mice that lack LEC-specific MHC-II expression, heterotopic tumor growth is attenuated, which is associated with increased numbers of tumor-specific CD8+ and effector CD4+ T cells, as well as decreased numbers of T regulatory CD4+ cells in the TME. Mechanistically, we show that murine and human dermal LECs can take up tumor antigens in vitro. Antigen-loaded LECs in vitro can induce antigen-specific proliferation of CD8+ T cells but not CD4+ T cells; however, these proliferated CD8+ T cells have reduced effector function in the presence of antigen-loaded LECs. Taken together, our study suggests LECs can act as immunosuppressive cells in the TME in an MHC-II dependent manner. Whether this is a result of direct tumor antigen presentation on MHC-II requires additional investigation.

Doxycycline for the treatment of breast cancer-related lymphedema.

Purpose: Secondary lymphedema is a common complication of cancer treatment for which no effective drug treatments yet exist. Level I clinical data suggests that doxycycline is effective for treating filariasis-induced lymphedema, in which it decreases tissue edema and skin abnormalities; however, this treatment has not been tested for cancer-related lymphedema. Over the past year, we used doxycycline in an off-label manner in patients with breast cancer-related secondary lymphedema. The purpose of this report was to retrospectively analyze the efficacy of this treatment. Methods: Patients who presented to our lymphedema clinic between January 2021 and January 2022 were evaluated, and barring allergies or contraindications to doxycycline treatment, were counseled on the off-label use of this treatment. Patients who wished to proceed were treated with doxycycline (200 mg given orally once daily) for 6 weeks. After IRB approval of this study, lymphedema outcomes were retrospectively reviewed. Results: Seventeen patients with a mean follow-up of 17.0 ± 13.2 weeks were identified in our retrospective review. Although doxycycline treatment had no significant effect on relative limb volume change or L-Dex scores, we found a significant improvement in patient-reported quality of life. Analysis of patient responses to the Lymphedema Life Impact Scale showed a significant improvement in the total impairment score due to improvements in the physical and psychological well-being subscales (p = 0.03, p = 0.03, p = 0.04, respectively). Conclusion: This small, retrospective study did not show significant improvements in limb volume or L-Dex scores in patients with breast cancer-related lymphedema treated with doxycycline. However, our patients reported improvements in quality-of-life measures using a validated lymphedema patient-reported outcome instrument. Our results suggest that doxycycline may be of use in patients with breast cancer-related lymphedema; however, larger and more rigorous studies are needed.

Adipokines in Sleep Disturbance and Metabolic Dysfunction: Insights from Network Analysis.

Adipokines are a growing group of secreted proteins that play important roles in obesity, sleep disturbance, and metabolic derangements. Due to the complex interplay between adipokines, sleep, and metabolic regulation, an integrated approach is required to better understand the significance of adipokines in these processes. In the present study, we created and analyzed a network of six adipokines and their molecular partners involved in sleep disturbance and metabolic dysregulation. This network represents information flow from regulatory factors, adipokines, and physiologic pathways to disease processes in metabolic dysfunction. Analyses using network metrics revealed that obesity and obstructive sleep apnea were major drivers for the sleep associated metabolic dysregulation. Two adipokines, leptin and adiponectin, were found to have higher degrees than other adipokines, indicating their central roles in the network. These adipokines signal through major metabolic pathways such as insulin signaling, inflammation, food intake, and energy expenditure, and exert their functions in cardiovascular, reproductive, and autoimmune diseases. Leptin, AMP activated protein kinase (AMPK), and fatty acid oxidation were found to have global influence in the network and represent potentially important interventional targets for metabolic and sleep disorders. These findings underscore the great potential of using network based approaches to identify new insights and pharmaceutical targets in metabolic and sleep disorders.

Structural and Functional Changes in Aged Skin Lymphatic Vessels.

Lymphatic structure and function play a critical role in fluid transport, antigen delivery, and immune homeostasis. A dysfunctional lymphatic system is associated with chronic low-grade inflammation of peripheral tissues, poor immune responses, and recurrent infections, which are also hallmarks of aging pathology. Previous studies have shown that aging impairs lymphatic structure and function in a variety of organ systems, including the intestines and central nervous system. However, previous studies are mostly limited to qualitative analysis of lymphatic structural changes and quantification of intestinal collecting vessel contractile function. It is not clear whether decreased lymphatic function contributes to pathological conditions related to aging, nor how it affects the skin immune microenvironment. Further, the effects of aging on skin initial and collecting lymphatic vessels, dendritic cell (DC) migration, cutaneous lymphatic pumping, and VEGFR-3 signaling in lymphatic endothelial cells (LECs) have not been quantitatively analyzed. Here, using fluorescent immunohistochemistry and flow cytometry, we confirm that aging decreases skin initial and collecting lymphatic vessel density. Indocyanine green (ICG) lymphangiography and DC migration assays confirm that aging decreases both fluid pumping and cell migration via lymphatic vessels. At the cellular level, aging causes decreased VEGFR-3 signaling, leading to increased LEC apoptosis and senescence. Finally, we determined that aging causes decreased lymphatic production of chemokines and alters LEC expression of junctional and adhesion molecules. This in turn leads to increased peri-lymphatic inflammation and nitrosative stress that might contribute to aging pathology in a feed-forward manner. Taken together, our study, in addition to quantitatively corroborating previous findings, suggests diverse mechanisms that contribute to lymphatic dysfunction in aging that in turn exacerbate the pathology of aging in a feed-forward manner.

Artificial Intelligence and Circulating Cell-Free DNA Methylation Profiling: Mechanism and Detection of Alzheimer's Disease.

Background: Despite extensive efforts, significant gaps remain in our understanding of Alzheimer's disease (AD) pathophysiology. Novel approaches using circulating cell-free DNA (cfDNA) have the potential to revolutionize our understanding of neurodegenerative disorders. Methods: We performed DNA methylation profiling of cfDNA from AD patients and compared them to cognitively normal controls. Six Artificial Intelligence (AI) platforms were utilized for the diagnosis of AD while enrichment analysis was used to elucidate the pathogenesis of AD. Results: A total of 3684 CpGs were significantly (adj. p-value < 0.05) differentially methylated in AD versus controls. All six AI algorithms achieved high predictive accuracy (AUC = 0.949−0.998) in an independent test group. As an example, Deep Learning (DL) achieved an AUC (95% CI) = 0.99 (0.95−1.0), with 94.5% sensitivity and specificity. Conclusion: We describe numerous epigenetically altered genes which were previously reported to be differentially expressed in the brain of AD sufferers. Genes identified by AI to be the best predictors of AD were either known to be expressed in the brain or have been previously linked to AD. We highlight enrichment in the Calcium signaling pathway, Glutamatergic synapse, Hedgehog signaling pathway, Axon guidance and Olfactory transduction in AD sufferers. To the best of our knowledge, this is the first reported genome-wide DNA methylation study using cfDNA to detect AD.

TGF-ß1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation.

BACKGROUND: Secondary lymphedema is a common complication of cancer treatment, and previous studies have shown that the expression of transforming growth factor-beta 1 (TGF-ß1), a pro-fibrotic and anti-lymphangiogenic growth factor, is increased in this disease. Inhibition of TGF-ß1 decreases the severity of the disease in mouse models; however, the mechanisms that regulate this improvement remain unknown. METHODS: Expression of TGF-ß1 and extracellular matrix molecules (ECM) was assessed in biopsy specimens from patients with unilateral breast cancer-related lymphedema (BCRL). The effects of TGF-ß1 inhibition using neutralizing antibodies or a topical formulation of pirfenidone (PFD) were analyzed in mouse models of lymphedema. We also assessed the direct effects of TGF-ß1 on lymphatic endothelial cells (LECs) using transgenic mice that expressed a dominant-negative TGF-ß receptor selectively on LECs (LECDN-RII ). RESULTS: The expression of TGF-ß1 and ECM molecules is significantly increased in BCRL skin biopsies. Inhibition of TGF-ß1 in mouse models of lymphedema using neutralizing antibodies or with topical PFD decreased ECM deposition, increased the formation of collateral lymphatics, and inhibited infiltration of T cells. In vitro studies showed that TGF-ß1 in lymphedematous tissues increases fibroblast, lymphatic endothelial cell (LEC), and lymphatic smooth muscle cell stiffness. Knockdown of TGF-ß1 responsiveness in LECDN-RII resulted in increased lymphangiogenesis and collateral lymphatic formation; however, ECM deposition and fibrosis persisted, and the severity of lymphedema was indistinguishable from controls. CONCLUSIONS: Our results show that TGF-ß1 is an essential regulator of ECM deposition in secondary lymphedema and that inhibition of this response is a promising means of treating lymphedema.

SARS-CoV-2 seroprevalence in the city of Hyderabad, India in early 2021.

Background: COVID-19 emerged as a global pandemic in 2020, spreading rapidly to most parts of the world. The proportion of infected individuals in a population can be reliably estimated via serosurveillance, making it a valuable tool for planning control measures. Our serosurvey study aimed to investigate SARS-CoV-2 seroprevalence in the urban population of Hyderabad at the end of the first wave of infections. Methods: This cross-sectional survey, conducted in January 2021 and including males and females aged 10 years and above, used multi-stage random sampling. 9363 samples were collected from 30 wards distributed over six zones of Hyderabad, and tested for antibodies against SARS-CoV-2 nucleocapsid antigen. Results: Overall seropositivity was 54.2%, ranging from 50% to 60% in most wards. Highest exposure appeared to be among those aged 30-39 and 50-59 years, with women showing greater seropositivity. Seropositivity increased with family size, with only marginal differences among people with varying levels of education. Seroprevalence was significantly lower among smokers. Only 11% of the survey subjects reported any COVID-19 symptoms, while 17% had appeared for COVID-19 testing. Conclusion: Over half the city's population was infected within a year of onset of the pandemic. However, ∼ 46% of people remained susceptible, contributing to subsequent waves of infection.