DJ-X-013 reduces LPS-induced inflammation, modulates Th17/ myeloid-derived suppressor cells, and alters NF-κB expression to ameliorate experimental colitis.

SCOPE: Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition of unknown etiology, although recent evidence suggests that it is caused by an excessive immune response to mucosal antigens. We determined the anti-inflammatory properties of novel compound DJ-X-013 in vitro in lipopolysaccharide (LPS)-induced macrophages and in an in vivo dextran sodium sulfate (DSS)-induced model of colitis. METHODS AND RESULTS: To evaluate the anti-inflammatory properties of DJ-X-013, we used LPS-activated RAW 264.7 macrophages in vitro and a DSS-induced experimental model of colitis in vivo. We examine cellular morphology, and tissue architecture by histology, flow cytometry, RT-qPCR, multiplex, and immunoblot analysis to perform cellular and molecular studies. DJ-X-013 treatment altered cell morphology and expression of inflammatory cytokines in LPS-activated macrophages as compared to cells treated with LPS alone. DJ-X-013 also impeded the migration of RAW 264.7 macrophages by modulating cytoskeletal organization and suppressed the expression of NF-κB and inflammatory markers as compared to LPS alone. DJ-X-013 treatment improved body weight, and colon length and attenuated inflammation in the colon of DSS-induced colitis. Intriguingly, DSS-challenged mice treated with DJ-X-013 induced the numbers of myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and natural killer T cells (NKT) in the colon lamina propria (LP) relative to DSS. DJ-X-013 also reduced the influx of neutrophils, TNF-α producing macrophages, restricted the number of Th17 cells, and suppressed inflammatory cytokines and NF-κB in the LP relative to DSS. CONCLUSION: DJ-X-013 is proposed to be a therapeutic strategy for ameliorating inflammation and experimental colitis.

Polyphenols: Role in Modulating Immune Function and Obesity.

Polyphenols, long-used components of medicinal plants, have drawn great interest in recent years as potential therapeutic agents because of their safety, efficacy, and wide range of biological effects. Approximately 75% of the world's population still use plant-based medicinal compounds, indicating the ongoing significance of phytochemicals for human health. This study emphasizes the growing body of research investigating the anti-adipogenic and anti-obesity functions of polyphenols. The functions of polyphenols, including phenylpropanoids, flavonoids, terpenoids, alkaloids, glycosides, and phenolic acids, are distinct due to changes in chemical diversity and structural characteristics. This review methodically investigates the mechanisms by which naturally occurring polyphenols mediate obesity and metabolic function in immunomodulation. To this end, hormonal control of hunger has the potential to inhibit pro-obesity enzymes such as pancreatic lipase, the promotion of energy expenditure, and the modulation of adipocytokine production. Specifically, polyphenols affect insulin, a hormone that is essential for regulating blood sugar, and they also play a role, in part, in a complex web of factors that affect the progression of obesity. This review also explores the immunomodulatory properties of polyphenols, providing insight into their ability to improve immune function and the effects of polyphenols on gut health, improving the number of commensal bacteria, cytokine production suppression, and immune cell mediation, including natural killer cells and macrophages. Taken together, continuous studies are required to understand the prudent and precise mechanisms underlying polyphenols' therapeutic potential in obesity and immunomodulation. In the interim, this review emphasizes a holistic approach to health and promotes the consumption of a wide range of foods and drinks high in polyphenols. This review lays the groundwork for future developments, indicating that the components of polyphenols and their derivatives may provide the answer to urgent worldwide health issues. This compilation of the body of knowledge paves the way for future discoveries in the global treatment of pressing health concerns in obesity and metabolic diseases.

Inhibition of microRNA-34c reduces detrusor ROCK2 expression and urinary bladder inflammation in experimental cystitis.

Interstitial cystitis (IC), also called painful bladder syndrome (PBS), is 2 to 5 times more common in women than in men, yet its cause and pathogenesis remain unclear. In our study using the cyclophosphamide (CYP)-induced mouse model of cystitis, histological evaluation of the urinary bladder (UB) lamina propria (LP) showed immune cell infiltrations, indicating moderate to severe inflammation. In this study, we noticed a differential expression of a subset of microRNAs (miRs) in the UB cells (UBs) of CYP-induced cystitis as compared to the control. UB inflammatory scores and inflammatory signaling were also elevated in CYP-induced cystitis as compared to control. We identified eight UBs miRs that exhibited altered expression after CYP induction and are predicted to have a role in inflammation and smooth muscle function (miRs-34c-5p, -34b-3p, -212-3p, -449a-5p, -21a-3p, -376b-3p, -376b-5p and - 409-5p). Further analysis using ELISA for inflammatory markers and real-time PCR (RT-PCR) for differentially enriched miRs identified miR-34c as a potential target for the suppression of UB inflammation in cystitis. Blocking miR-34c by antagomir ex vivo reduced STAT3, TGF-ß1, and VEGF expression in the UBs, which was induced during cystitis as compared to control. Interestingly, miR-34c inhibition also downregulated ROCK2 but elevated ROCK1 expression in bladder and detrusor cells. Thus, the present study shows that targeting miR-34c can mitigate the STAT3, TGF-ß, and VEGF, inflammatory signaling in UB, and suppress ROCK2 expression in UBs to effectively suppress the inflammatory response in cystitis. This study highlights miR-34c as a potential biomarker and/or serves as the basis for new therapies for the treatment of cystitis.

miR-10a-3p modulates adiposity and suppresses adipose inflammation through TGF-ß1/Smad3 signaling pathway.

Background: Obesity is a multifactorial disease characterized by an enhanced amount of fat and energy storage in adipose tissue (AT). Obesity appears to promote and maintain low-grade chronic inflammation by activating a subset of inflammatory T cells, macrophages, and other immune cells that infiltrate the AT. Maintenance of AT inflammation during obesity involves regulation by microRNAs (miRs), which also regulate the expression of genes implicated in adipocyte differentiation. This study aims to use ex vivo and in vitro approaches to evaluate the role and mechanism of miR-10a-3p in adipose inflammation and adipogenesis. Methods: Wild-type BL/6 mice were placed on normal (ND) and high-fat diet (HFD) for 12 weeks and their obesity phenotype, inflammatory genes, and miRs expression were examined in the AT. We also used differentiated 3T3-L1 adipocytes for mechanistic in vitro studies. Results: Microarray analysis allowed us to identify an altered set of miRs in the AT immune cells and Ingenuity pathway analysis (IPA) prediction demonstrated that miR-10a-3p expression was downregulated in AT immune cells in the HFD group as compared to ND. A molecular mimic of miR-10a-3p reduced expression of inflammatory M1 macrophages, cytokines, and chemokines, including transforming growth factor-beta 1 (TGF-ß1), transcription factor Krüppel-like factor 4 (KLF4), and interleukin 17F (IL-17F) and induced expression of forkhead box P3 (FoxP3) in the immune cells isolated from AT of HFD-fed mice as compared to ND. In differentiated 3T3-L1 adipocytes, the miR-10a-3p mimics also reduced expression of proinflammatory genes and lipid accumulation, which plays a role in the dysregulation of AT function. In these cells, overexpression of miR-10a-3p reduced the expression of TGF-ß1, Smad3, CHOP-10, and fatty acid synthase (FASN), relative to the control scramble miRs. Conclusion: Our findings suggest that miR-10a-3p mimic mediates the TGF-ß1/Smad3 signaling to improve metabolic markers and adipose inflammation. This study provides a new opportunity for the development of miR-10a-3p as a novel therapeutic for adipose inflammation, and its associated metabolic disorders.

Piceatannol induces regulatory T cells and modulates the inflammatory response and adipogenesis.

The beneficial effects of the polyphenolic compound piceatannol (PC) has been reported for metabolic diseases, antiproliferative, antioxidant, and anti-cancer properties. Despite its beneficial effects on inflammatory diseases, little is known about how PC regulates inflammatory responses and adipogenesis. Therefore, this study was designed to determine the effects of PC on the inflammatory response and adipogenesis. The effect of PC on splenocytes, 3T3-L1 adipocytes, and RAW264.7 macrophages was analyzed by flow cytometry, qRT-PCR, morphometry, and western blot analysis. PC induced apoptosis in activated T cells in a dose-dependent manner using stimulated splenocytes and reduced the activation of T cells, altered T cell frequency, and interestingly induced the frequency of regulatory T (Treg) cells as compared to controls. PC suppressed the expression of TNF-α, iNOS, IL-6R, and NF-κB activation in RAW264.7 macrophages after lipopolysaccharides (LPS)-induction as compared to the control. Interestingly, PC altered the cell morphology of 3T3-L1 adipocytes with a concomitant decrease in cell volume, lipid deposition, and TNF-α expression, but upregulation of leptin and IL-1ß. Our findings suggested that PC induced apoptosis in activated T cells, decreased immune cell activation and inflammatory response, and hindered adipogenesis. This new set of data provides promising hope as a new therapeutic to treat both inflammatory disease and obesity.

MicroRNAs: a crossroad that connects obesity to immunity and aging.

Obesity is characterized by an elevated amount of fat and energy storage in the adipose tissue (AT) and is believed to be the root cause of many metabolic diseases (MDs). Obesity is associated with low-grade chronic inflammation in AT. Like obesity, chronic inflammation and MDs are prevalent in the elderly. The resident immune microenvironment is not only responsible for maintaining AT homeostasis but also plays a crucial role in stemming obesity and related MDs. Mounting evidence suggests that obesity promotes activation in resident T cells and macrophages. Additionally, inflammatory subsets of T cells and macrophages accumulated into the AT in combination with other immune cells maintain low-grade chronic inflammation. microRNAs (miRs) are small non-coding RNAs and a crucial contributing factor in maintaining immune response and obesity in AT. AT resident T cells, macrophages and adipocytes secrete various miRs and communicate with other cells to create a potential effect in metabolic organ crosstalk. AT resident macrophages and T cells-associated miRs have a prominent role in regulating obesity by targeting several signaling pathways. Further, miRs also emerged as important regulators of cellular senescence and aging. To this end, a clear link between miRs and longevity has been demonstrated that implicates their role in regulating lifespan and the aging process. Hence, AT and circulating miRs can be used as diagnostic and therapeutic tools for obesity and related disorders. In this review, we discuss how miRs function as biomarkers and impact obesity, chronic inflammation, and aging.

Pathophysiological relationship between hypoxia associated oxidative stress, Epithelial-mesenchymal transition, stemness acquisition and alteration of Shh/ Gli-1 axis during oral sub-mucous fibrosis and oral squamous cell carcinoma.

Oral sub-mucous fibrosis (OSF) is a pathophysiological state of oral cavity or oropharynx having a high chance of conversion to oral squamous cell carcinoma (OSCC). It involves fibrotic transformation of sub-epithelial matrix along with epithelial abnormalities. The present work aims to unveil the mechanistic domain regarding OSF to OSCC conversion exploring the scenario of hypoxia associated oxidative stress, epithelial-mesenchymal transition (EMT), metastasis and stemness acquisition. The study involves histopathological analysis of the diseased condition along with the exploration of oxidative stress status, assessment of mitochondrial condition, immunohistochemical analysis of HIF-1α, E-cadherin, vimentin, ERK, ALDH-1, CD133, Shh, Gli-1 and survivin expressions in the oral epithelial region together with the quantitative approach towards collagen deposition in the sub-epithelial matrix. Oxidative stress was found to be associated with type-II EMT in case of OSF attributing the development of sub-epithelial fibrosis and type-III EMT in case of OSCC favoring malignancy associated metastasis. Moreover, the acquisition of stemness during OSCC can also be correlated with EMT. Alteration of Shh and Gli-1 expression pattern revealed the mechanistic association of hypoxia with the phenotypic plasticity and disease manifestation in case of OSF as well as OSCC. Shh/ Gli-1 signaling can also be correlated with survivin mediated cytoprotective phenomenon under oxidative stress. Overall, the study established the correlative network of hypoxia associated oxidative stress, EMT and manifestation of oral pre-cancerous and cancerous condition in a holistic approach that may throw rays of hope in the therapeutic domain of the concerned diseases.

Attenuation corrected-optical coherence tomography for quantitative assessment of skin wound healing and scar morphology.

Imaging the structural modifications of underlying tissues is vital to monitor wound healing. Optical coherence tomography (OCT) images high-resolution sub-surface information, but suffers a loss of intensity with depth, limiting quantification. Hence correcting the attenuation loss is important. We performed swept source-OCT of full-thickness excision wounds for 300 days in mice skin. We used single-scatter attenuation models to determine and correct the attenuation loss in the images. The phantom studies established the correspondence of corrected-OCT intensity (reflectivity) with matrix density and hydration. We histologically validated the corrected-OCT and measured the wound healing rate. We noted two distinct phases of healing-rapid and steady-state. We also detected two compartments in normal scars using corrected OCT that otherwise were not visible in the OCT scans. The OCT reflectivity in the scar compartments corresponded to distinct cell populations, mechanical properties and composition. OCT reflectivity has potential applications in evaluating the therapeutic efficacy of healing and characterizing scars.

Structural mechanics modeling reveals stress-adaptive features of cutaneous scars.

The scar is a predominant outcome of adult mammalian wound healing despite being associated with partial function loss. Here in this paper, we have described the structure of a full-thickness normal scar as a "di-fork" with dual biomechanical compartments using in vivo and ex vivo experiments. We used structural mechanics simulations to model the deformation fields computationally and stress distribution in the scar in response to external forces. Despite its loss of tissue components, we have found that the scar has stress-adaptive features that cushion the underlying tissues from external mechanical impacts. Thus, this new finding can motivate research to understand the biomechanical advantages of a scar in maintaining the primary function of the skin, i.e., mechanical barrier despite permanent loss of some tissues and specialized functions.

Impact of intercellular connectivity on epithelial mesenchymal transition plasticity.

Epithelial mesenchymal transition (EMT) in development, tissue repair and carcinogenesis involves cellular plasticity with varying degrees of epithelial and mesenchymal properties. Several recent studies have focused on EMT phenotypic dynamism; however, information on cellular interaction in the context of EMT is inadequate. In our previous study, we investigated EMT phenotypic plasticity and anticipated it as a population driven interactive process. Present study has characterized cellular connectivity as a representative of interactivity during EMT in epithelial normal and cancer cell. It has also explored dynamism of connectivity and phenotype employing Markov model. Further, plasticity was substantiated with cell surface microvilli and molecular marker. The study unveiled interplay between phenotype and connectivity too. Findings have revealed that intercellular connectivity fueled EMT plasticity and its dynamism was more prominent in cancer population. However, normal cells are more vibrant in transition and phenotypic plasticity. We have proposed connectivity plasticity as a hallmark of EMT and needs to be studied in depth. Present study also paves the way in translating in vitro EMT findings in histopathological practices.

Analysis of Carcinogenic Heavy Metals in Gallstones and its Role in Gallbladder Carcinogenesis.

PURPOSE: Gallstone is a high-risk factor for gallbladder pre-malignancy or malignancy (GB PM-M) but which substances of gallstones definitely assist to turn out in to GB PM-M, remains unclear. This study aimed to find out the presence of carcinogenic heavy metals in gallstones and to explore the aetiopathogenesis of gallbladder pre-malignancy and malignancy. METHODS: Presence of elements in gallstones was detected by energy dispersive X-ray spectroscopy (EDS) with scanning electron microscopy (SEM) and then level of carcinogenic heavy metals was estimated in gallstones using atomic absorption spectroscopy (AAS). The experiment was carried out in gallstone samples of 46 patients with gallbladder pre-malignant and malignant condition (PM-M group) and 65 sex and age-matched patients with chronic cholecystitis (C-C group). Gallstones were also classified in to three types such as cholesterol stone, mixed stone, and black pigment stone. RESULTS: EDS analysis detected presence of mercury, lead, and cobalt elements in all types of gallstones of both PM-M and C-C groups. AAS analysis revealed significantly higher amount of mercury (p < 0.001), lead (p < 0.0001), cobalt (p < 0.01), and cadmium (p < 0.01) in the gallstones of PM-M than C-C groups. The presence of these heavy metals also varied among stone types of both groups. EDS phase analysis showed 'dense deposits' of these metals in gallstones. CONCLUSIONS: Presence of significantly higher amount of mercury, lead, cobalt, and cadmium in gallstones may play a pivotal role as risk factors in the development of gallbladder malignancy or pre-malignancy. 'Dense deposits' of these metals in the gallstones which is the first observation, may act as crucial doses of carcinogens.

Modeling continuum of epithelial mesenchymal transition plasticity.

Living systems respond to ambient pathophysiological changes by altering their phenotype, a phenomenon called 'phenotypic plasticity'. This program contains information about adaptive biological dynamism. Epithelial-mesenchymal transition (EMT) is one such process found to be crucial in development, wound healing, and cancer wherein the epithelial cells with restricted migratory potential develop motile functions by acquiring mesenchymal characteristics. In the present study, phase contrast microscopy images of EMT induced HaCaT cells were acquired at 24 h intervals for 96 h. The expression study of relevant pivotal molecules viz. F-actin, vimentin, fibronectin and N-cadherin was carried out to confirm the EMT process. Cells were intuitively categorized into five distinct morphological phenotypes. A population of 500 cells for each temporal point was selected to quantify their frequency of occurrence. The plastic interplay of cell phenotypes from the observations was described as a Markovian process. A model was formulated empirically using simple linear algebra, to depict the possible mechanisms of cellular transformation among the five phenotypes. This work employed qualitative, semi-quantitative and quantitative tools towards illustration and establishment of the EMT continuum. Thus, it provides a newer perspective to understand the embedded plasticity across the EMT spectrum.

Modulating prime molecular expressions and in vitro wound healing rate in keratinocyte (HaCaT) population under characteristic honey dilutions.

ETHNOPHARMACOLOGY RELEVANCE: In traditional medicines honey is known for healing efficacy and vividly used as "Anupan" in Ayurvedic medicines appreciating roles in dilutions. Validating efficacy of physico-chemically characterized honey in dilutions, studies on in vitro wound healing and attainment of cellular confluence epithelial cells including expressions of cardinal genes is crucial. To evaluate effects of characterized honey in varied dilutions on cellular viability, in vitro wound healing and modulation of prime epithelial gene expressions. MATERIALS AND METHODS: Six Indian honey-samples from different sources were physico-chemically characterized and optimal one was explored in dilutions (v/v%) through in vitro studies on human epithelial (HaCaT) cells for viability, wound healing and expressions of genes p63, E-cadherin, ß-catenin, GnT-III and GnT-V. RESULTS: Studied honey samples (i.e. A-F) depicted range of pH (2-4), water (12.48-23.95), electrical conductivity (2.57-14.34), carbohydrate (68.73-98.65), protein (.316-5.36) and antioxidant potential. Though sample A and F showed physico-chemical proximity, but overall bio-impact of the earlier was better, thus studied in 8-.1% (v/v) dilution range. Four dilutions (.01, .04, .1, .25 v/v%) augmented cellular viability but in vitro wound healing was fastest (p<.05) under .1%. Such efficacy was further documented for p63 up-regulation by immunocytochemistry and mRNA studies. The E-cadherin and ß-catenin mRNA-expressions were also up-regulated and their proteins were predominantly cytoplasmic. E-cadherin up-regulation was corroborative with down-regulation and up-regulation of GnT-III and GnT-V respectively. CONCLUSION: Present study illustrated efficacy of particular honey dilution (.1%) with characteristic free radical scavenging activity in facilitating cell proliferation and attainment of confluence towards faster wound healing and modulation of cardinal epithelial genes (viz. p63, E-cadherin, ß-catenin, Gnt-III and V).

The first cross sectional survey on intracranial malignancy in Kolkata, India: reflection of the state of the art in southern West Bengal.

Recent increase in the occurrence of intracranial malignancies and poor performance of therapeutic measures have established the disease as an important concern of medical sciences. The lack of information about the disease pattern throughout India creates problems for maintaining community health for prevention. The present study on the hospital population of Kolkata was conducted to determine the incidence pattern of the disease in the population of southern West Bengal, focusing on distribution with age, sex, occupation and religion in different districts of the region, and characterizing diagnostic and therapeutic measures. Among a total of 39,509 cancer patients from 21 health centers of Kolkata, 2.4% had brain cancers and among these more than 60% are gliomas. A cross-sectional study for a period of 3 years reported the occurrence of 15 types of intracranial malignancy, which demonstrated astrocytomas (36.8%), glioblastoma multiforme (GBM) (7.9%) and meningiomas (11.6%) to be predominant. Brain tumors occur more frequently in males with few exceptions and the incidence was found to be highest among the 40-49 year old group (20.2%). No specific trend for religion and occupation was apparent. However, the district wise distribution showed maximum incidences among industrial areas, namely, Kolkata (33.1%), North 24-Parganas (18.2%), Howrah (9.3%) and Hoogly (7.6%). Diagnosis of the disease was by CT scan, MRI and histological identification (pre and post operative). Therapeutic procedures rely mainly on surgery and radiotherapy, whereas chemotherapy was used as an adjuvant for about 10% of the cases. Evaluation of the scenario regarding intracranial malignancy in this region was a long awaited requirement which should ultimately serve an important function in pointing to risk zones within the population and allow better control measures to be introduced for the disease.