OBJECTIVE

Myxomatous mitral valve disease (MMVD) is associated with leaflet thickening, fibrosis, matrix remodelling, and leaflet prolapse. Molecular mechanisms contributing to MMVD, however, remain poorly understood. We tested the hypothesis that increased transforming growth factor-β (TGF-β) signalling and reactive oxygen species (ROS) are major contributors to pro-fibrotic gene expression in human and mouse mitral valves.

RESULTS

Using qRT-PCR, we found that increased expression of TGF-β1 in mitral valves from humans with MMVD (n = 24) was associated with increased expression of connective tissue growth factor (CTGF) and matrix metalloproteinase 2 (MMP2). Increased levels of phospho-SMAD2/3 (western blotting) and expression of SMAD-specific E3 ubiquitin-protein ligases (SMURF) 1 and 2 (qRT-PCR) suggested that TGF-β1 signalling occurred through canonical signalling cascades. Oxidative stress (dihydroethidium staining) was increased in human MMVD tissue and associated with increases in NAD(P)H oxidase catalytic subunits (Nox) 2 and 4, occurring despite increases in superoxide dismutase 1 (SOD1). In mitral valves from SOD1-deficient mice, expression of CTGF, MMP2, Nox2, and Nox4 was significantly increased, suggesting that ROS can independently activate pro-fibrotic and matrix remodelling gene expression patterns. Furthermore, treatment of mouse mitral valve interstitial cells with cell permeable antioxidants attenuated TGF-β1-induced pro-fibrotic and matrix remodelling gene expression in vitro.

CONCLUSIONS

Activation of canonical TGF-β signalling is a major contributor to fibrosis and matrix remodelling in MMVD, and is amplified by increases in oxidative stress. Treatments aimed at reducing TGF-β activation and oxidative stress in early MMVD may slow progression of MMVD.

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different proinflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic (IA) injections of media (control) or Ureaplasma parvum serovar 3 either 7 or 70 d before preterm delivery. Another group received an IA injection of Escherichia coli LPS 2 d prior to delivery. To test for interactions, IA U. parvum-exposed animals were challenged with IA LPS and delivered 2 d later. All animals were delivered at 124 ± 1-d gestation (term = 150 d). Compared with the 2-d LPS exposure group, the U. parvum 70 d + LPS group had 1) decreased lung pro- and anti-inflammatory cytokine expression and 2) fewer CD3(+) T lymphocytes, CCL2(+), myeloperoxidase(+), and PU.1(+) cells in the lung. Interestingly, exposure to U. parvum for 7 d did not change responses to a subsequent IA LPS challenge, and exposure to IA U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGF-β1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70 d + LPS but not U. parvum 7 d + LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of downregulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis-exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

BACKGROUND

Hepatology research has focused on developing traditional therapies as pharmacological medicines to treat liver cirrhosis. Thus, this study evaluated mechanisms of the hepatoprotective activity of Curcuma longa rhizome ethanolic extract (CLRE) on thioacetamide-induced liver cirrhosis in rats.

METHODS

The hepatoprotective effect of CLRE was measured in a rat model of thioacetamide-induced liver cirrhosis over 8 weeks. Hepatic cytochrome P450 2E1 and serum levels of TGF-β1 and TNF-α were evaluated. Oxidative stress was measured by malondialdehyde, urinary 8-hydroxyguanosine and nitrotyrosine levels. The protective activity of CLRE free-radical scavenging mechanisms were evaluated through antioxidant enzymes. Protein expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins in animal blood sera was studied and confirmed by immunohistochemistry of Bax, Bcl2 proteins and proliferating cell nuclear antigen.

RESULTS

Histopathology, immunohistochemistry and liver biochemistry were significantly lower in the Curcuma longa-treated groups compared with controls. CLRE induced apoptosis, inhibited hepatocytes proliferation but had no effect on hepatic CYP2E1 levels.

CONCLUSIONS

The progression of liver cirrhosis could be inhibited by the antioxidant and anti-inflammatory activities of CLRE and the normal status of the liver could be preserved.

Liver fibrosis is a common pathway leading to cirrhosis and a worldwide clinical issue. Astaxanthin is a red carotenoid pigment with antioxidant, anticancer, and anti-inflammatory properties. The aim of this study was to investigate the effect of astaxanthin on liver fibrosis and its potential protective mechanisms. Liver fibrosis was induced in a mouse model using CCL4 (intraperitoneal injection, three times a week for 8 weeks), and astaxanthin was administered everyday at three doses (20, 40, and 80 mg/kg). Pathological results indicated that astaxanthin significantly improved the pathological lesions of liver fibrosis. The levels of alanine aminotransferase aspartate aminotransferase and hydroxyproline were also significantly decreased by astaxanthin. The same results were confirmed in bile duct liagtion, (BDL) model. In addition, astaxanthin inhibited hepatic stellate cells (HSCs) activation and formation of extracellular matrix (ECM) by decreasing the expression of NF-κB and TGF-β1 and maintaining the balance between MMP2 and TIMP1. In addition, astaxanthin reduced energy production in HSCs by downregulating the level of autophagy. These results were simultaneously confirmed in vivo and in vitro. In conclusion, our study showed that 80 mg/kg astaxanthin had a significant protective effect on liver fibrosis by suppressing multiple profibrogenic factors.

Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial-mesenchymal transition (EMT), TGF-β1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM(-/-)) wounds. Correspondingly, VIM(-/-) wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM(-/-) wounds. Vimentin reconstitution in VIM(-/-) fibroblasts restored both their proliferation and TGF-β1 production. Similarly, restoring paracrine TGF-β-Slug-EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-β1-Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation.

BACKGROUND

Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results.

OBJECTIVE

In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration.

METHODS

L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1β (IL-1β) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers.

RESULTS

More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-β1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1β between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-β1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1β on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001).

CONCLUSIONS

In comparison to L-PRP, L-PRF had higher amounts of released TGF-β1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model.

CONCLUSIONS

By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.

Matrix metalloproteinase-9 (MMP-9) is one of the major components of the matrix proteolytic network, and its role in the pathogenesis of renal interstitial fibrosis remains largely unknown. Here, we demonstrate that ablation of MMP-9 attenuated renal interstitial fibrotic lesions in obstructive nephropathy. Mice lacking MMP-9 were less likely to develop morphological injury, which was characterized by a reduced disruption of tubular basement membrane (TBM) and expression of fibronectin as well as deposition of total tissue collagen in the kidneys after sustained ureteral obstruction compared with their wild-type counterparts. Deficiency of MMP-9 blocked tubular epithelial-to-myofibroblast transition (EMT) but did not alter the induction of transforming growth factor (TGF)-β1 axis expression in the obstructed kidneys. In vitro, TBM, which was digested by MMP-9 instead of MMP-9 itself, induces EMT and enhances migration of transformed cells. Thus increased MMP-9 is detrimental in renal interstitial fibrogenesis through a cascade of events that leads to TBM destruction and in turn to promotion of EMT. Our findings establish a crucial and definite importance of MMP-9 in the pathogenesis of renal interstitial fibrosis at the whole-animal level.

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

BACKGROUND

TGF-β signaling has been extensively studied in many developmental contexts, amongst which is its ability to induce epithelial to mesenchymal transitions (EMT). EMTs play crucial roles during embryonic development and have also come under intense scrutiny as a mechanism through which breast cancers progress to become metastatic. Interestingly, while the molecular hallmarks of EMT progression (loss of cell adhesion, nuclear localization of β-catenin) are straightforward, the cellular signaling cascades that result in an EMT are numerous and diverse. Furthermore, most studies describing the biological effects of TGF-β have been performed using high concentrations of active, soluble TGF-β, despite the fact that TGF-β is produced and secreted as a latent complex.

METHODS

MCF-7 breast cancer cells treated with recombinant IGF-1 were assayed for metalloproteinase activity and invasiveness through a matrigel coated transwell invasion chamber. IGF-1 treatments were then followed by the addition of latent-TGF-β1 to determine if elevated levels of IGF-1 together with latent-TGF-β1 could cause EMT.

RESULTS

Results showed that IGF-1 - a molecule known to be elevated in breast cancer is a regulator of matrix metalloproteinase activity (MMP) and the invasive potential of MCF-7 breast cancer cells. The effects of IGF-1 appear to be mediated through signals transduced via the PI3K and MAPK pathways. In addition, increased IGF-1, together with latent TGF-β1 and active MMPs result in EMT.

CONCLUSIONS

Taken together our data suggest a novel a link between IGF-1 levels, MMP activity, TGF-β signaling, and EMT in breast cancer cells.

Eggs of the helminth Schistosoma mansoni accumulate in the colon following infection and generate Th2-biassed inflammatory granulomas which become down- modulated in size as the infection proceeds to chronicity. However, although CD4+CD25+FoxP3+ regulatory T cells (T(regs)) are known to suppress Th1-mediated colitis, it is not clear whether they control Th2-associated pathologies of the large intestine which characterise several helminth infections. Here we used a novel 3D-multiphoton confocal microscopy approach to visualise and quantify changes in the size and composition of colonic granulomas at the acute and chronic phases of S. mansoni infection. We observed decreased granuloma size, as well as reductions in the abundance of DsRed+ T cells and collagen deposition at 14 weeks (chronic) compared to 8 weeks (acute) post-infection. Th2 cytokine production (i.e. IL-4, IL-5) in the colonic tissue and draining mesenteric lymph node (mLN) decreased during the chronic phase of infection, whilst levels of TGF-β1 increased, co-incident with reduced mLN proliferative responses, granuloma size and fibrosis. The proportion of CD4+CD25+FoxP3+T(regs): CD4+ cells in the mLN increased during chronic disease, while within colonic granulomas there was an approximate 4-fold increase. The proportion of CD4+CD25+FoxP3+T(regs) in the mLN that were CD103+ and CCR5+ also increased indicating an enhanced potential to home to intestinal sites. CD4+CD25+ cells suppressed antigen-specific Th2 mLN cell proliferation in vitro, while their removal during chronic disease resulted in significantly larger granulomas, partial reversal of Th2 hypo-responsiveness and an increase in the number of eosinophils in colonic granulomas. Finally, transfer of schistosome infection-expanded CD4+CD25+T(regs) down-modulated the development of colonic granulomas, including collagen deposition. Therefore, CD4+CD25+FoxP3+T(regs) appear to control Th2 colonic granulomas during chronic infection, and are likely to play a role in containing pathology during intestinal schistosomiasis.

Mesenchymal stem cell (MSC) is a promising tool for the therapy of immune disorders. However, their efficacy and mechanisms in treating allergic skin disorders are less verified. We sought to investigate the therapeutic efficacy of human umbilical cord blood-derived MSCs (hUCB-MSCs) against murine atopic dermatitis (AD) and to explore distinct mechanisms that regulate their efficacy. AD was induced in mice by the topical application of Dermatophagoides farinae. Naïve or activated-hUCB-MSCs were administered to mice, and clinical severity was determined. The subcutaneous administration of nucleotide-binding oligomerization domain 2 (NOD2)-activated hUCB-MSCs exhibited prominent protective effects against AD, and suppressed the infiltration and degranulation of mast cells (MCs). A β-hexosaminidase assay was performed to evaluate the effect of hUCB-MSCs on MC degranulation. NOD2-activated MSCs reduced the MC degranulation via NOD2-cyclooxygenase-2 signaling. In contrast to bone marrow-derived MSCs, hUCB-MSCs exerted a cell-to-cell contact-independent suppressive effect on MC degranulation through the higher production of prostaglandin E2 (PGE2 ). Additionally, transforming growth factor (TGF)-β1 production from hUCB-MSCs in response to interleukin-4 contributed to the attenuation of MC degranulation by downregulating FcεRI expression in MCs. In conclusion, the subcutaneous application of NOD2-activated hUCB-MSCs can efficiently ameliorate AD, and MSC-derived PGE2 and TGF-β1 are required for the inhibition of MC degranulation.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced collagen-rich fibrosis known as desmoplastic reaction; however, the role of fibrosis in PDAC is poorly understood. In this report we show that collagen can regulate the tumor suppressive let-7 family of microRNAs in pancreatic cancer cells. PDAC cells growing in 3D collagen gels repress mature let-7 without affecting the precursor form of let-7 in part through increased expression of membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) and ERK1/2 activation. PDAC cells in collagen also demonstrate increased TGF-β1 signaling, and blocking TGF-β1 signaling attenuated collagen-induced MT1-MMP expression, ERK1/2 activation and repression of let-7 levels. Although MT1-MMP overexpression was not sufficient to inhibit let-7 on 2D tissue culture plastic, overexpression of MT1-MMP in PDAC cells embedded in 3D collagen gels or grown in vivo repressed let-7 levels. Importantly, MT1-MMP expression significantly correlated with decreased levels of let-7 in human PDAC tumor specimens. Overall, our study emphasizes the interplay between the key proteinase MT1-MMP and its substrate type I collagen in modulating microRNA expression, and identifies an additional mechanism by which fibrosis may contribute to PDAC progression.

GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4(+) T cells results in lack of expression of latent TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-β1/GARP complex on their cell surface rather than by secreted latent TGF-β1.

BACKGROUND

MicroRNAs have been demonstrated to play an important role in the pathogenesis of diabetic nephropathy (DN). In this study, we investigated both the repertoire of miRNAs in the kidneys of patients with DN and their potential regulatory role in inflammation-mediated glomerular endothelial injury.

METHODS

The miRNA expression profiling of the renal biopsy samples was performed by a microarray analysis; then, in situ hybridization and real-time polymerase chain reaction (PCR) were used to determine the localization and expression of two of the miRNAs significantly up-regulated in human DN kidney samples, miR-155 and miR-146a, in the kidney tissues from type 1 and type 2 DN rat models. Human renal glomerular endothelial cells (HRGECs) cultured under high-glucose conditions were transfected with miR-155 and miR-146a mimics, and the transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, and nuclear factor (NF)-κB expressions were examined by western blot, real-time PCR, and an electrophoresis mobility shift assay.

RESULTS

The expression of both miR-155 and miR-146a was increased more than fivefold in the kidney samples of the DN patients compared with the controls, and the miR-155 expression was closely correlated with the serum creatinine levels (R = 0.95, P = 0.004). During the induction and progression of the disease in type 1 and type 2 DN rat models, miR-155 and miR-146a were demonstrated to increase gradually. In vitro, high glucose induced the over-expression of miR-155 and miR-146a in the HRGECs, which, in turn, increased the TNF-α, TGF-β1, and NF-κB expression.

CONCLUSIONS

Taken together, these findings indicate that the increased expression of miR-155 and miR-146a in the DN patients and in the experimental DN animal models was found to contribute to inflammation-mediated glomerular endothelial injury.

MicroRNAs (miRs) seem to mediate renal fibrosis in several renal diseases, with some miRs having profibrotic effects and others having opposing effects. Although differential expression of certain miRs has been described in lupus nephritis, it is unknown whether miRs contribute to fibrosis or could serve as biomarkers of specific histologic manifestations of lupus nephritis. Here, we compared miR expression in kidney biopsies from patients with lupus nephritis and identified miR-150 as the most differentially expressed miR in kidneys with high chronicity (chronicity index [CI] ≥ 4); miR-150 positively correlated with chronicity scores and the expression of profibrotic proteins. Overexpression of miR-150 significantly reduced expression of the antifibrotic protein suppressor of cytokine signaling 1 (SOCS1) and upregulated profibrotic proteins in both proximal tubular and mesangial cells. Directly targeting SOCS1 with a small interfering RNA produced similar results. Furthermore, TGF-β1 induced miR-150 expression, decreased SOCS1, and increased profibrotic proteins in proximal tubular cells and podocytes; a miR-150 inhibitor reversed these changes, suggesting that the profibrotic effects of TGF-β1 are, at least in part, mediated by miR-150. Consistent with these in vitro observations, biopsies with high miR-150 and high CI exhibited substantial expression of TGF-β1, reduced SOCS1, and an increase in profibrotic proteins. In summary, miR-150 is a promising quantitative renal biomarker of kidney injury in lupus nephritis. Our results suggest that miR-150 promotes renal fibrosis by increasing profibrotic molecules through downregulation of SOCS1.

Cerebral small-vessel disease is a common disorder in elderly populations; however, its molecular basis is not well understood. We recently demonstrated that mutations in the high-temperature requirement A (HTRA) serine peptidase 1 (HTRA1) gene cause a hereditary cerebral small-vessel disease, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HTRA1 belongs to the HTRA protein family, whose members have dual activities as chaperones and serine proteases and also repress transforming growth factor-β (TGF-β) family signaling. We demonstrated that CARASIL-associated mutant HTRA1s decrease protease activity and fail to decrease TGF-β family signaling. However, the precise molecular mechanism for decreasing the signaling remains unknown. Here we show that increased expression of ED-A fibronectin is limited to cerebral small arteries and is not observed in coronary, renal arterial or aortic walls in patients with CARASIL. Using a cell-mixing assay, we found that HTRA1 decreases TGF-β1 signaling triggered by proTGF-β1 in the intracellular space. HTRA1 binds and cleaves the pro-domain of proTGF-β1 in the endoplasmic reticulum (ER), and cleaved proTGF-β1 is degraded by ER-associated degradation. Consequently, the amount of mature TGF-β1 is reduced. These results establish a novel mechanism for regulating the amount of TGF-β1, specifically, the intracellular cleavage of proTGF-β1 in the ER.

OBJECTIVE

To investigate intrathecal immune activation parameters and HIV-RNA in HIV-associated neurocognitive disorders (HAND) of advanced naïve HIV-infected patients and to evaluate their dynamics before and after initiation of antiretroviral therapy (ART).

METHODS

Cross-sectional and longitudinal analysis of HIV RNA, proinflammatory cytokines (IL-6, IL-10, INF-γ, TNF-α, TGF-β1, and TGF-β2) and chemokines (MIP-1α, MIP-1β, and MCP-1) in plasma and cerebrospinal fluid (CSF) of HIV-infected patients with CD4 <200/μL.

RESULTS

HAND was diagnosed at baseline in 6/12 patients. Baseline CSF HIV-RNA was comparable in patients with or without HAND, whereas CSF concentration of IL-6 and MIP-1β, proinflammatory cytokines, was increased in HAND patients. CSF evaluation at 12 weeks was available in 10/12 cases. ART greatly reduced HIV-RNA in all patients. Nevertheless, IL-6 and MIP-1β remained elevated after 12 weeks of therapy in HAND patients, in whom CSF HIV RNA decay was slower than the plasmatic one as well.

CONCLUSIONS

Immune activation, as indicated by inflammatory cytokines, but not higher levels of HIV-RNA is observed in advanced naïve HIV-infected patients with HAND. In HAND patients, ART introduction resulted in a less rapid clearance of CSF viremia compared to plasma and no modifications of intratechal immune activation.

OBJECTIVE

To investigate the plasma dynamics of 5 proinflammatory/fibrogenic cytokines, including interleukin-1beta (IL-1β), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta1 (TGF-β1) to ascertain their value in predicting radiation-induced lung toxicity (RILT), both individually and in combination with physical dosimetric parameters.

METHODS

Treatments of patients receiving definitive conventionally fractionated radiation therapy (RT) on clinical trial for inoperable stages I-III lung cancer were prospectively evaluated. Circulating cytokine levels were measured prior to and at weeks 2 and 4 during RT. The primary endpoint was symptomatic RILT, defined as grade 2 and higher radiation pneumonitis or symptomatic pulmonary fibrosis. Minimum follow-up was 18 months.

RESULTS

Of 58 eligible patients, 10 (17.2%) patients developed RILT. Lower pretreatment IL-8 levels were significantly correlated with development of RILT, while radiation-induced elevations of TGF-ß1 were weakly correlated with RILT. Significant correlations were not found for any of the remaining 3 cytokines or for any clinical or dosimetric parameters. Using receiver operator characteristic curves for predictive risk assessment modeling, we found both individual cytokines and dosimetric parameters were poor independent predictors of RILT. However, combining IL-8, TGF-ß1, and mean lung dose into a single model yielded an improved predictive ability (P<.001) compared to either variable alone.

CONCLUSIONS

Combining inflammatory cytokines with physical dosimetric factors may provide a more accurate model for RILT prediction. Future study with a larger number of cases and events is needed to validate such findings.

Background: Colorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Exosome shave emerged as crucial regulators of intercellular communication and that abundant Circular RNAs (circRNAs) are enriched within exosomes. CircRNAs are novel members of noncoding RNAs regulating cancer proliferation and progression. However, the function and regulatory mechanism of cancer-derived exosomal circRNAs in CRC remains unclear.

Methods: CRC cells-derived exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot. CCK-8, wound healing and transwell assays, and flow cytometry assays were conducted to assess whether exosomes would affect the proliferation, metastasis, and apoptosis of CRC cells, respectively. Moreover, we performed the RNA sequencing and RT-qPCR to identify circRNAs in exosome-stimulated CRC cells. Fluorescence in situ hybridization (FISH) assay was used to detect the cellular distribution of circPACRGL. Bioinformatic analyses (StarBase 2.0) were used to pool the miRNA targets of circPACRGL. Luciferase assays were performed to verify the direct interaction. Finally, flow cytometry was used to detect the differentiation of N1-N2 neutrophils.

Results: Our study identified a novel CRC-derived exosomal circRNA, circPACRGL. We found circPACRGL was significantly upregulated in CRC cells after tumor-derived exosomes addition. Moreover, circPACRGL serves as a sponge for miR-142-3p/miR-506-3p to facilitate the transforming growth factor-β1 (TGF-β1) expression. As a result, circPACRGL promoted CRC cell proliferation, migration and invasion, as well as differentiation of N1 to N2 neutrophils via miR-142-3p/miR-506-3p-TGF-β1 axis.

Conclusion: Our study, the first to reveal that cancer-derived exosomal circPACRGL plays an oncogenic role in CRC proliferation and metastasis, providing mechanistic insights into the roles of circRNAs in CRC progression and a valuable marker for CRC treatment.

Keywords: Colorectal cancer; Exosome; Invasion; Migration; TGF-β1; circPACRGL; miR-142-3p; miR-506-3p.

B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-β1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.

The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects.

Dysregulation of microRNAs has been implicated in many cardiovascular diseases including fibrosis. Here we report that miR-433 was consistently elevated in three models of heart disease with prominent cardiac fibrosis, and was enriched in fibroblasts compared to cardiomyocytes. Forced expression of miR-433 in neonatal rat cardiac fibroblasts increased proliferation and their differentiation into myofibroblasts as determined by EdU incorporation, α-SMA staining, and expression levels of fibrosis-associated genes. Conversely, inhibition of miR-433 exhibited opposite results. AZIN1 and JNK1 were identified as two target genes of miR-433. Decreased level of AZIN1 activated TGF-β1 while down-regulation of JNK1 resulted in activation of ERK and p38 kinase leading to Smad3 activation and ultimately cardiac fibrosis. Importantly, systemic neutralization of miR-433 or adeno-associated virus 9 (AAV9)-mediated cardiac transfer of a miR-433 sponge attenuated cardiac fibrosis and ventricular dysfunction following myocardial infarction. Thus, our work suggests that miR-433 is a potential target for amelioration of cardiac fibrosis.

BACKGROUND

Recent findings suggest that BPH has an inflammatory component. Clinical trials have documented that therapy with LHRH antagonist Cetrorelix causes a marked and prolonged improvement in LUTS in men with symptomatic BPH. We investigated the mechanism of action and effect of Cetrorelix in a rat model of BPH.

METHODS

Adult male Wistar rats were used. BPH was induced in rats by subcutaneous injections of TE 2 mg/day for 4 weeks. Control animals received injections of corn oil. After induction of BPH, rats received depot Cetrorelix pamoate at the doses of 0.625, 1.25, and 12.5 mg/kg on days 1 and 22 and TE-control rats received vehicle injections. Whole prostates were weighed and processed for RNA and protein. Real-time RT-PCR assays for numerous inflammatory cytokines and growth factors were performed. Quantitative analyses of prostatic LHRH receptor, LHRH, androgen receptor (AR) and 5α-reductase 2 were done by real-time RT-PCR and immunoblotting; serum DHT, LH, PSA, and IGF-1 by immunoassays.

RESULTS

mRNA levels for inflammatory cytokines IFN-γ, IL-3, IL-4, IL-5, IL-6, IL-8, IL-13, IL-15, and IL-17 and for growth factors EGF, FGF-2, FGF-7, FGF-8, FGF-14, TGF-β1, and VEGF-A were significantly reduced by Cetrorelix 0.625 mg/kg (P < 0.05). Prostate weights were also significantly lowered by any dose of Cetrorelix.

CONCLUSIONS

This study suggests that Cetrorelix reduces various inflammatory cytokines and growth factors in rat prostate and, at doses which do not induce castration levels of testosterone, can lower prostate weights. Our findings shed light on the mechanism of action of LHRH antagonists in BPH.

Galectin-9 (Gal-9) ameliorates autoimmune reactions by suppressing Th17 cells while augmenting Foxp3(+) regulatory T cells (Tregs). However, the exact mechanism of Gal-9-mediated immune modulation has been elusive. In a MOG-induced experimental allergic encephalomyelitis model using Gal-9(-/-) mice, we observed exacerbated inflammation and an increase in IL-17-producing Th17 cells balanced by a decrease in Foxp3+ Tregs. During in vitro Th17 skewing using TGF-β1 and IL-6, exogenous Gal-9 suppressed Th17 cell development and expanded Foxp3(+) Tregs from naïve CD4 T cells in an IL-2-dependent manner. Although Gal-9 induced cell death in Tim3-expressing differentiated Th17 cells, Gal-9 suppressed Th17 development in a Tim-3-independent. Benzyl-α-GalNAc (an O-glycan biosynthesis inhibitor), but not swainsonine (a complex-type N-glycan biosynthesis inhibitor) abrogated Gal-9-mediated inhibition of Th17 development indicating that there is a linkage between Gal-9 and an unidentified glycoprotein(s) with O-linked β-galactosides that suppress Th17 development.