Journal of Clinical Medicine

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Open AccessFeature PaperReview

Mechanisms of Regulatory B cell Function in Autoimmune and Inflammatory Diseases beyond IL-10

by Avijit Ray and Bonnie N. Dittel

J. Clin. Med. 2017, 6(1), 12; https://doi.org/10.3390/jcm6010012 - 23 Jan 2017

Cited by 62 | Viewed by 6955

In the past two decades it has become clear that in addition to antigen presentation and antibody production B cells play prominent roles in immune regulation. While B cell-derived IL-10 has garnered much attention, B cells also effectively regulate inflammation by a variety [...] Read more.

In the past two decades it has become clear that in addition to antigen presentation and antibody production B cells play prominent roles in immune regulation. While B cell-derived IL-10 has garnered much attention, B cells also effectively regulate inflammation by a variety of IL-10-independent mechanisms. B cell regulation has been studied in both autoimmune and inflammatory diseases. While collectively called regulatory B cells (Breg), no definitive phenotype has emerged for B cells with regulatory potential. This has made their study challenging and thus unique B cell regulatory mechanisms have emerged in a disease-dependent manner. Thus to harness the therapeutic potential of Breg, further studies are needed to understand how they emerge and are induced to evoke their regulatory activities. Full article

(This article belongs to the Special Issue B Cells in Autoimmunity)

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Open AccessFeature PaperReview

TGF-? Signaling in Gastrointestinal Cancers: Progress in Basic and Clinical Research

by Takehiko Yokobori and Masahiko Nishiyama

J. Clin. Med. 2017, 6(1), 11; https://doi.org/10.3390/jcm6010011 - 18 Jan 2017

Cited by 34 | Viewed by 8461

Abstract

Transforming growth factor (TGF)-? superfamily proteins have many important biological functions, including regulation of tissue differentiation, cell proliferation, and migration in both normal and cancer cells. Many studies have reported that TGF-? signaling is associated with disease progression and therapeutic resistance in several [...] Read more.

Transforming growth factor (TGF)-? superfamily proteins have many important biological functions, including regulation of tissue differentiation, cell proliferation, and migration in both normal and cancer cells. Many studies have reported that TGF-? signaling is associated with disease progression and therapeutic resistance in several cancers. Similarly, TGF-?-induced protein (TGFBI)—a downstream component of the TGF-? signaling pathway—has been shown to promote and/or inhibit cancer. Here, we review the state of basic and clinical research on the roles of TGF-? and TGFBI in gastrointestinal cancers. Full article

(This article belongs to the Special Issue Biological and Clinical Aspects of TGF-beta in Carcinogenesis)

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Open AccessFeature PaperArticle

Integrated Treatment of PTSD and Substance Use Disorders: The Mediating Role of PTSD Improvement in the Reduction of Depression

by Kristina J. Korte, Kaitlin E. Bountress, Rachel L. Tomko, Therese Killeen, Megan Moran-Santa Maria and Sudie E. Back

J. Clin. Med. 2017, 6(1), 9; https://doi.org/10.3390/jcm6010009 - 13 Jan 2017

Cited by 8 | Viewed by 9500

Abstract

Posttraumatic stress disorder (PTSD) represents one of the most common mental health disorders, particularly among veterans, and is associated with significant distress and impairment. This highly debilitating disorder is further complicated by common comorbid psychiatric disorders, such as substance use disorders (SUD). Individuals [...] Read more.

Posttraumatic stress disorder (PTSD) represents one of the most common mental health disorders, particularly among veterans, and is associated with significant distress and impairment. This highly debilitating disorder is further complicated by common comorbid psychiatric disorders, such as substance use disorders (SUD). Individuals with PTSD and co-occurring SUD also commonly present with secondary symptoms, such as elevated depression. Little is known, however, about how these secondary symptoms are related to treatment outcome. The aim of the present study, therefore, was to examine (1) the effects of treatment of comorbid PTSD/SUD on depressive symptoms; and (2) whether this effect was mediated by changes in PTSD severity or changes in SUD severity. Participants were 81 U.S. military veterans (90.1% male) with PTSD and SUD enrolled in a randomized controlled trial examining the efficacy of an integrated, exposure-based treatment (Concurrent Treatment of PTSD and Substance Use Disorders Using Prolonged Exposure; n = 54) versus relapse prevention (n = 27). Results revealed significantly lower depressive symptoms at post-treatment in the COPE group, as compared to the relapse prevention group. Examination of the mechanisms associated with change in depression revealed that reduction in PTSD severity, but not substance use severity, mediated the association between the treatment group and post-treatment depression. The findings underscore the importance of treating PTSD symptoms in order to help reduce co-occurring symptoms of depression in individuals with PTSD/SUD. Clinical implications and avenues for future research are discussed. Full article

(This article belongs to the Special Issue Post-Traumatic Stress Disorder)

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Open AccessReview

Mammary Gland Involution Provides a Unique Model to Study the TGF-? Cancer Paradox

by Qiuchen Guo, Courtney Betts, Nathan Pennock, Elizabeth Mitchell and Pepper Schedin

J. Clin. Med. 2017, 6(1), 10; https://doi.org/10.3390/jcm6010010 - 13 Jan 2017

Cited by 27 | Viewed by 10539

Abstract

Transforming Growth Factor-? (TGF-?) signaling in cancer has been termed the “TGF-? paradox”, acting as both a tumor suppresser and promoter. The complexity of TGF-? signaling within the tumor is context dependent, and greatly impacted by cellular crosstalk between TGF-? responsive cells in [...] Read more.

Transforming Growth Factor-? (TGF-?) signaling in cancer has been termed the “TGF-? paradox”, acting as both a tumor suppresser and promoter. The complexity of TGF-? signaling within the tumor is context dependent, and greatly impacted by cellular crosstalk between TGF-? responsive cells in the microenvironment including adjacent epithelial, endothelial, mesenchymal, and hematopoietic cells. Here we utilize normal, weaning-induced mammary gland involution as a tissue microenvironment model to study the complexity of TGF-? function. This article reviews facets of mammary gland involution that are TGF-? regulated, namely mammary epithelial cell death, immune activation, and extracellular matrix remodeling. We outline how distinct cellular responses and crosstalk between cell types during physiologically normal mammary gland involution contribute to simultaneous tumor suppressive and promotional microenvironments. We also highlight alternatives to direct TGF-? blocking anti-cancer therapies with an emphasis on eliciting concerted microenvironmental-mediated tumor suppression. Full article

(This article belongs to the Special Issue Biological and Clinical Aspects of TGF-beta in Carcinogenesis)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Rodent model of synchronized weaning-induced mammary gland involution. Male and female rodents are mated for 3–5 days. After pup birth, litter size is normalized to ensure equal lactation load per dam, and weaning initiated during peak lactation (9–13 days) by pup removal. The kinetics of three major cellular events during involution are shown in the above timeline: epithelial cell apoptosis, immune cell influx, and extracellular matrix remodeling. Days since start of lactation are indicated by “D” followed by a number, and days since synchronized weaning by “Inv”. Whole gland representations of mammary epithelial ducts (black lines) as well as milk accumulation (white filled circles) are displayed below the corresponding developmental stages in the timeline. Full article ">Figure 2
The tumor suppressive and promotional functions of transforming growth factor β (TGF-β) in the involuting mammary microenvironment. Each panel depicts an involuting mammary acini either lacking (top panel) or containing (bottom panel) tumor cells. (A) Epithelium: In non-transformed mammary epithelial cells (top half of diagram, blue arrows), TGF-β suppresses cell proliferation, and induces tumor suppressive apoptosis and phagocytosis mediated by loss of epithelial junctions. In the presence of transformed cells (bottom half of diagram, red arrows), TGF-β can promote cancer progression by inducing epithelial mesenchymal transition (EMT) and stem cell phenotypes. Additionally, anti-proliferative functions of TGF-β can be lost in tumor cells via mutations in TGF-β signaling pathways (depicted by red X); (B) Immune milieu: In the absence of tumor cells (top half of diagram, blue arrows), TGF-β suppresses chronic inflammation by inducing T-helper 2 (Th2) cells and T-helper 17 (Th17) cells which can suppress T-helper 1 (Th1) cells mediated tumor initiation. This immune environment also maintains epithelial stem cell health and epithelial cell junctional integrity (blue arrows). In the presence of tumor cells (bottom half of diagram, red arrows), TGF-β induced Th2 immunity suppresses anti-cancer CD8 T cell cytotoxic function and directly activates tumor cells through growth factor/cytokine signaling; (C) Extracellular matrix/fibroblast: Active TGF-β is released in the extracellular microenvironment when proteases cleave the Latency Associated Peptide (LAP). TGF-β signaling within fibroblasts impairs production of stromal cell-derived factor-1 (SDF1). In the absence of tumor, TGF-β signaling plays a critical role in maintaining tissue integrity (top half of diagram, blue arrows). In the presence of tumor cells (bottom half of diagram, red arrows) a wound healing like extracellular matrix environment provides stratum and accompanying signals for cancer cell motility and invasive phenotypes. Full article ">Figure 3
Immune cells increase in the mammary gland during involution. An influx of immune cells consistent with classic wound healing is observed during mammary gland involution including various myeloid cell populations (left axis): Macrophages and monocytes (CD45+ Gr1intermediate/low F480+ CD11b+, red line); dendritic cells and macrophages (CD45+ CD11c+ MHCII+, black line); and granulocytes (CD45+ Gr1high F480− CD11b+, green line). T cells are also increased in the mammary gland during involution (right axis): CD4 T cells (CD45+ CD3+ CD4+, blue line), CD8 T cells (CD45+ CD3+ CD8+, gray line) and the immunosuppressive Treg T cells (CD45+ CD3+ CD4+ FoxP3+ CD25+, orange line). On the X-axis, the involution window is labeled in red, as “Inv” followed by a number for the day post-weaning. Both axes represent frequencies of indicated cell populations as a fraction of total cells from the gland, as determined by single cell suspensions analyzed by flow cytometry. The Figure is derived from data reported in Reference [<a href="#B45-jcm-06-00010" class="html-bibr">45</a>]. Full article ">Figure 4
Tissue-level TGF-β signaling and implications for TGF-β targeted therapy. TGF-β can simultaneously mediate pro-tumor (red arrows) and anti-tumor (blue arrows) activities, depending on responding cell type. The effect of an anti-TGF-β therapy would be expected to result in both dampened pro-tumor (red dotted lines) and dampened anti-tumor (blue dotted lines) activities of TGF-β, resulting in mixed tumor outcome. Anti-tumor effects of blocking TGF-β are decreased tumor cell proliferation, M2 polarization, and extracellular matrix (ECM) deposition, and pro-tumorigenic effects include pro-inflammatory tumor initiation (green cell) and enhanced fibroblast production of pro-tumor stromal cell-derived factor 1 (SDF1) and TGF-β. An alternative strategy to direct TGF-β targeting may be to target downstream effectors. TGF-β is known to induce cyclooxygenase-2 (COX2) and subsequent prostaglandin E2 (PGE2) (green circle) production by a number of cell types. Targeting COX2 activity (processes depicted by green arrows) targets pathways in the established cancer environment that may have more uni-directionally pro-tumor effects, such as M2/Th2 polarization and ECM deposition, without impairing TGF-β mediated tumor suppressive functions. Full article ">

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Open AccessEditorial

Acknowledgement to Reviewers of Journal of Clinical Medicine in 2016

by Journal of Clinical Medicine Editorial Office

J. Clin. Med. 2017, 6(1), 8; https://doi.org/10.3390/jcm6010008 - 11 Jan 2017

Cited by 3 | Viewed by 4378

Abstract

The editors of Journal of Clinical Medicine would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2016.[...] Full article

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Open AccessFeature PaperReview

Stromal Modulators of TGF-? in Cancer

by Brunella Costanza, Ijeoma Adaku Umelo, Justine Bellier, Vincent Castronovo and Andrei Turtoi

J. Clin. Med. 2017, 6(1), 7; https://doi.org/10.3390/jcm6010007 - 6 Jan 2017

Cited by 138 | Viewed by 22437

Abstract

Transforming growth factor-? (TGF-?) is an intriguing cytokine exhibiting dual activities in malignant disease. It is an important mediator of cancer invasion, metastasis and angiogenesis, on the one hand, while it exhibits anti-tumor functions on the other hand. Elucidating the precise role of [...] Read more.

Transforming growth factor-? (TGF-?) is an intriguing cytokine exhibiting dual activities in malignant disease. It is an important mediator of cancer invasion, metastasis and angiogenesis, on the one hand, while it exhibits anti-tumor functions on the other hand. Elucidating the precise role of TGF-? in malignant development and progression requires a better understanding of the molecular mechanisms involved in its tumor suppressor to tumor promoter switch. One important aspect of TGF-? function is its interaction with proteins within the tumor microenvironment. Several stromal proteins have the natural ability to interact and modulate TGF-? function. Understanding the complex interplay between the TGF-? signaling network and these stromal proteins may provide greater insight into the development of novel therapeutic strategies that target the TGF-? axis. The present review highlights our present understanding of how stroma modulates TGF-? activity in human cancers. Full article

(This article belongs to the Special Issue Biological and Clinical Aspects of TGF-beta in Carcinogenesis)

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Canonical and non-canonical TGF-β signaling pathways. (A) In the canonical signaling pathway, biologically active TGF-β ligands bind to TGFβRII, which in turn activates TGFβRI. TGFβRI-regulated SMAD2/3 proteins are phosphorylated at their C-terminal serine residues and form complexes with SMAD4 (co-SMAD), initiating a number of biological processes through transcriptional regulation of target genes. (B) In the non-canonical signaling pathways, the TGF-β receptor complex transmits its signal through other factors, such as the mitogen-activated protein kinases (MAPKs), phosphatidylinositide 3-kinase (PI3K), TNF receptor-associated factor 4/6 (TRAF4/6) and Rho family of small GTPases. Activated MAPKs can exert transcriptional regulation either through direct interaction with the nuclear SMAD protein complex or via other downstream proteins. Moreover, activated JNK/p38/ERK act in concert with SMADs to regulate cellular apoptosis and proliferation, whereas they mediate metastasis, angiogenesis and cellular growth through other transcription factors, such as c-JUN and ATF. RhoA/ROCK can be activated by TGF-β to induce actin stress fiber formation during EMT via a non-transcriptional mechanism. TGF-β can activate PI3K and AKT by inducing a physical interaction between the PI3K p85 subunit and the receptor complex leading to translational responses via mTOR/S6kinase activation. TGF-β activation of the TRAF proteins can initiate nuclear factor-κB (NF-κB) signaling activity, leading to the inflammatory response among other processes. The arrows indicate activation/signaling direction of the respective pathway. Full article ">Figure 2
TGF-β-mediated cancer cell/stromal cell crosstalk. (A) TGF-β can activate resident stromal cells giving rise to cancer-associated fibroblasts (CAFs). In cancer cells, TGF-β promotes the transcription of SNAIL, the functional loss of E-cadherin, the acquisition of an EMT phenotype and the recruitment of SMAD/AKT signaling proteins. The process of metastasis is further supported by activated CAFs through secretion of IL-11 or IL-6, which further promotes STAT3 signaling in cancer cells. (B) TGF-β can trigger angiogenesis in endothelial cells through activation of VEGFR2 by VEGF. The TGF-β-mediated angiogenic effect on cancer cells is regulated by TGFβRII/SMAD3-dependent upregulation of fibroblast growth factor-2 (FGF2) expression and release in the stroma. (C) Cancer cells via the induction of aberrant TGF-β signaling can induce the down-regulation of CAV1 in adjacent fibroblasts leading to a CAF phenotype. The loss of CAV1 has been observed to lead to an increase in oxidative stress, activation of HIF-1α and the induction of aerobic glycolysis. Under these conditions, CAF have been reported to produce and secrete lactate, which is used as fuel by cancer cells. Blue arrows indicate proteins secreted by cancer cells. Magenta arrows indicate proteins secreted by stromal cells. Black arrows indicate overexpression (upward pointing) and down-regulation (downward pointing) of target proteins. Full article ">Figure 3
Stromal activators of TGF-β in the tumor microenvironment. (A) MT1-MMP, MMP2 and MMP9 proteolytically cleave latent transforming growth factor-β binding protein (LTBP), thereby releasing latent TGF-β from the extracellular matrix. Plasmin, thrombin, BMP1 and fibulin-2 also activate TGF-β through cleavage or interaction with LTBP1. (B) MMP2, MMP3, MMP9 and MMP13 activate latent TGF-β via proteolytic cleavage of the latency-associated peptide (LAP), while integrins expressed on fibroblasts (αvβ3, αvβ5 and αvβ8) bind to the large latent complex (LLC) and activate latent TGF-β through MT1-MMP-dependent cleavage of LAP. (C) Integrins αvβ-1 and 5 bind to the LLC and induce conformational changes in the latent complex via contractile action from activated fibroblasts. (D) ROS produced by activated fibroblasts via the induction of oxidative stress from adjacent cancer cells can lead to the oxidation of the LAP domain and induce allosteric changes that release mature TGF-β from LAP. The loss of CAV1 expression in activated fibroblasts is also associated with enhanced oxidative stress and increased production of ROS. (E) Thrombospondin-1 (TSP-1) directly interacts with the LAP domain, inducing conformational rearrangement of LAP and altering the interaction of LAP with the mature domain of TGF-β. The mature (active) form of TGF-β can then bind to its cognate receptor and exert its tumor promoting and tumor suppressive properties. Dashed arrow indicates recruitment of the mature TGF-β protein to its cognate receptor. Full article ">Figure 4
Stromal inhibitors of TGF-β in the tumor microenvironment. (A) The SLRPs, decorin, biglycan, lumican and asporin, bind with high affinity to TGF-β, preventing the biologically-active protein from binding to its cognate receptor. (B) Fibulin-3 and -4indirectly inhibit TGF-β activity by interacting with TGF-β RI, leading to a decrease in TGFβRI/TGFβRII complex formation. (C) Decorin can indirectly disrupt TGF-β activity by negative regulation of SMAD2 phosphorylation. (D) Nephrocan indirectly regulates TGF-β activity by inhibiting canonical SMAD3 signaling. (E) Fibromodulin and fibrilin-1 and -2 bind to the TGF-β LLC, preventing its release from the ECM. Fibronectin mediates the regulation of LTBP1 in the ECM, thereby suppressing TGF-β1 bioavailability. Solid arrows indicate activation and/or enzymatic activity while blunted arrows indicate inhibitory activity (solid—direct; interrupted—indirect activity). Full article ">

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Open AccessFeature PaperReview

Bronchopulmonary Dysplasia: Chronic Lung Disease of Infancy and Long-Term Pulmonary Outcomes

by Lauren M. Davidson and Sara K. Berkelhamer

J. Clin. Med. 2017, 6(1), 4; https://doi.org/10.3390/jcm6010004 - 6 Jan 2017

Cited by 306 | Viewed by 28074

Abstract

Bronchopulmonary dysplasia (BPD) is a chronic lung disease most commonly seen in premature infants who required mechanical ventilation and oxygen therapy for acute respiratory distress. While advances in neonatal care have resulted in improved survival rates of premature infants, limited progress has been [...] Read more.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease most commonly seen in premature infants who required mechanical ventilation and oxygen therapy for acute respiratory distress. While advances in neonatal care have resulted in improved survival rates of premature infants, limited progress has been made in reducing rates of BPD. Lack of progress may in part be attributed to the limited therapeutic options available for prevention and treatment of BPD. Several lung-protective strategies have been shown to reduce risks, including use of non-invasive support, as well as early extubation and volume ventilation when intubation is required. These approaches, along with optimal nutrition and medical therapy, decrease risk of BPD; however, impacts on long-term outcomes are poorly defined. Characterization of late outcomes remain a challenge as rapid advances in medical management result in current adult BPD survivors representing outdated neonatal care. While pulmonary disease improves with growth, long-term follow-up studies raise concerns for persistent pulmonary dysfunction; asthma-like symptoms and exercise intolerance in young adults after BPD. Abnormal ventilatory responses and pulmonary hypertension can further complicate disease. These pulmonary morbidities, combined with environmental and infectious exposures, may result in significant long-term pulmonary sequalae and represent a growing burden on health systems. Additional longitudinal studies are needed to determine outcomes beyond the second decade, and define risk factors and optimal treatment for late sequalae of disease. Full article

(This article belongs to the Special Issue Chronic Respiratory Diseases)

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Open AccessArticle

Combined Effects of Androgen and Growth Hormone on Osteoblast Marker Expression in Mouse C2C12 and MC3T3-E1 Cells Induced by Bone Morphogenetic Protein

by Kosuke Kimura, Tomohiro Terasaka, Nahoko Iwata, Takayuki Katsuyama, Motoshi Komatsubara, Ryota Nagao, Kenichi Inagaki and Fumio Otsuka

J. Clin. Med. 2017, 6(1), 6; https://doi.org/10.3390/jcm6010006 - 5 Jan 2017

Cited by 7 | Viewed by 7972

Abstract

Osteoblasts undergo differentiation in response to various factors, including growth factors and steroids. Bone mass is diminished in androgen- and/or growth hormone (GH)-deficient patients. However the functional relationship between androgen and GH, and their combined effects on bone metabolism, remains unclear. Here we [...] Read more.

Osteoblasts undergo differentiation in response to various factors, including growth factors and steroids. Bone mass is diminished in androgen- and/or growth hormone (GH)-deficient patients. However the functional relationship between androgen and GH, and their combined effects on bone metabolism, remains unclear. Here we investigated the mutual effects of androgen and GH on osteoblastic marker expression using mouse myoblastic C2C12 and osteoblast-like MC3T3-E1 cells. Combined treatment with dihydrotestosterone (DHT) and GH enhanced BMP-2-induced expression of Runx2, ALP, and osteocalcin mRNA, compared with the individual treatments in C2C12 cells. Co-treatment with DHT and GH activated Smad1/5/8 phosphorylation, Id-1 transcription, and ALP activity induced by BMP-2 in C2C12 cells but not in MC3T3-E1 cells. The insulin-like growth factor (IGF-I) mRNA level was amplified by GH and BMP-2 treatment and was restored by co-treatment with DHT in C2C12 cells. The mRNA level of the IGF-I receptor was not significantly altered by GH or DHT, while it was increased by IGF-I. In addition, IGF-I treatment increased collagen-1 mRNA expression, whereas blockage of endogenous IGF-I activity using an anti-IGF-I antibody failed to suppress the effect of GH and DHT on BMP-2-induced Runx2 expression in C2C12 cells, suggesting that endogenous IGF-I was not substantially involved in the underlying GH actions. On the other hand, androgen receptor and GH receptor mRNA expression was suppressed by BMP-2 in both cell lines, implying the existence of a feedback action. Collectively the results showed that the combined effects of androgen and GH facilitated BMP-2-induced osteoblast differentiation at an early stage by upregulating BMP receptor signaling. Full article

(This article belongs to the Special Issue Bone Cell Biology and Transcriptional Regulation)

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Graphical abstract

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Full article ">Figure 1
Effects of androgen on BMP-induced osteoblast marker expression. C2C12 cells (2 × 105 cells/well) were treated with the indicated concentrations of DHT, GH, and BMP-2 in serum-free DMEM. After 48-h culture, total cellular RNA was extracted and mRNA levels of Runx2, ALP, osteocalcin, osterix, collagen-1, and Id-1 were examined by real-time RT-PCR. The expression levels of target genes were standardized by RPL19 mRNA levels in each sample. Results in all panels are shown as means ± SEM of data from at least three separate experiments, each performed with triplicate samples. The results were analyzed by ANOVA. For each result within a panel, the values with different superscript letters are significantly different at p < 0.05. Full article ">Figure 2
Combined effects of androgen and GH on BMP-induced osteoblast marker expression. (A) C2C12 and (B) MC3T3-E1 cells (2 × 105 cells/well) were treated with the indicated concentrations of DHT, GH, and BMP-2 in a serum-free medium. After 48-h culture, total cellular RNA was extracted and mRNA levels of Id-1 were examined by real-time RT-PCR. The expression levels of target genes were standardized by RPL19 mRNA levels in each sample. Results in all panels are shown as means ± SEM of data from at least three separate experiments, each performed with triplicate samples. The results were analyzed by ANOVA. For each result within a panel, the values with different superscript letters are significantly different at p < 0.05. Full article ">Figure 3
Effects of androgen and GH on BMP-receptor signal activation. (A) C2C12 cells (1 × 105 cells/well) were precultured in a serum-free condition in the absence or presence of DHT and GH for 48 h and were then stimulated with BMP-2. After 1-h culture with BMP-2 treatment, cells were lysed and subjected to SDS-PAGE/immunoblotting (IB) analysis using antibodies that can detect pSmad1/5/8 and tSmad1. The integrated signal density of each protein band was digitally analyzed, and the ratios of signal intensities of pSmad/tSmad1 were calculated; (B) C2C12 and MC3T3-E1 cells (2 × 105 cells/well) were treated with the indicated concentrations of DHT, GH, and BMP-2 in a serum-free medium. After 48-h culture, total cellular RNA was extracted and mRNA levels of Id-1 were examined by real-time RT-PCR. The expression levels of the target gene were standardized by RPL19 mRNA levels in each sample; (C) C2C12 and MC3T3-E1 cells (1 × 105 cells/well) were treated with the indicated concentrations of BMP-2, DHT, and GH in a serum-free medium for 72 h, and then ALP activity in the cell lysates was determined. Results in all panels are shown as means ± SEM of data from at least three separate experiments, each performed with triplicate samples. The results were analyzed by ANOVA. For each result within a panel, the values with different superscript letters are significantly different at p < 0.05. Full article ">Figure 4
Involvement of IGF-I in osteoblast marker expression induced by BMP-2, androgen, and GH. (A,B) C2C12 and MC3T3-E1 cells (2 × 105 cells/well) were treated with the indicated concentrations of DHT, GH, and BMP-2 in a serum-free medium. After 48-h culture, total cellular RNA was extracted and mRNA levels of IGF-I and IGF-IR were examined by real-time RT-PCR. The expression levels of target genes were standardized by RPL19 mRNA levels in each sample; (C) After C2C12 cells (2 × 105 cells/well) had been treated with the indicated concentrations of IGF-I and anti-IGF-I IgG in a serum-free condition for 48 h, total cellular RNA was extracted and mRNA levels of IGF-IR and collagen-1 were examined by real-time RT-PCR. The expression levels of target genes were standardized by RPL19 mRNA levels in each sample; (D) BMP-2, DHT and GH were added to the cell culture in combination with either normal or anti-IGF-I IgG (1 μg/mL) in a serum-free condition for 48 h. The expression levels of Runx2 were determined by real-time RT-PCR and the expression levels of Runx2 mRNA were standardized by RPL19 mRNA levels in each sample. Results in all panels are shown as means ± SEM of data from at least three separate experiments, each performed with triplicate samples. The results were analyzed by ANOVA and the unpaired t-test. For each result within a panel, the values with different superscript letters are significantly different at p < 0.05 (A,B). * p < 0.05 vs. control group (C). n.s., not significant. Full article ">Figure 5
Mutual effects of androgen and GH on BMP-induced osteoblast marker expression. (A) C2C12 and (B) MC3T3-E1 cells (2 × 105 cells/well) were treated with the indicated concentrations of DHT, GH, and BMP-2 in a serum-free medium. After 48-h culture, total cellular RNA was extracted and mRNA levels of GHR and AR were examined by real-time RT-PCR. The expression levels of target genes were standardized by RPL19 mRNA levels in each sample. Results in all panels are shown as means ± SEM of data from at least three separate experiments, each performed with triplicate samples. The results were analyzed by ANOVA. For each result within a panel, the values with different superscript letters are significantly different at p < 0.05. Full article ">Figure 6
Interaction of androgen and GH in BMP-induced osteoblast marker expression. Combined treatment with DHT and GH enhanced BMP-2-induced expression of osteoblast markers. DHT and GH augmented BMP-2-induced Smad1/5/8 signaling and Id-1 transcription in C2C12 cells. The IGF-I mRNA level was amplified by GH and BMP-2, and the effect was reversed by DHT. The expression of AR and GHR was suppressed by BMP-2, suggesting a possible feedback loop. Full article ">

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Open AccessReview

The TGF-?/Smad4 Signaling Pathway in Pancreatic Carcinogenesis and Its Clinical Significance

by Sunjida Ahmed, Azore-Dee Bradshaw, Shweta Gera, M. Zahidunnabi Dewan and Ruliang Xu

J. Clin. Med. 2017, 6(1), 5; https://doi.org/10.3390/jcm6010005 - 5 Jan 2017

Cited by 136 | Viewed by 14790

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human cancers due to its complicated genomic instability. PDAC frequently presents at an advanced stage with extensive metastasis, which portends a poor prognosis. The known risk factors associated with PDAC include advanced age, [...] Read more.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human cancers due to its complicated genomic instability. PDAC frequently presents at an advanced stage with extensive metastasis, which portends a poor prognosis. The known risk factors associated with PDAC include advanced age, smoking, long-standing chronic pancreatitis, obesity, and diabetes. Its association with genomic and somatic mutations is the most important factor for its aggressiveness. The most common gene mutations associated with PDAC include KRas2, p16, TP53, and Smad4. Among these, Smad4 mutation is relatively specific and its inactivation is found in more than 50% of invasive pancreatic adenocarcinomas. Smad4 is a member of the Smad family of signal transducers and acts as a central mediator of transforming growth factor beta (TGF-?) signaling pathways. The TGF-? signaling pathway promotes many physiological processes, including cell growth, differentiation, proliferation, fibrosis, and scar formation. It also plays a major role in the development of tumors through induction of angiogenesis and immune suppression. In this review, we will discuss the molecular mechanism of TGF-?/Smad4 signaling in the pathogenesis of pancreatic adenocarcinoma and its clinical implication, particularly potential as a prognostic factor and a therapeutic target. Full article

(This article belongs to the Special Issue Biological and Clinical Aspects of TGF-beta in Carcinogenesis)

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Open AccessReview

Liquid Biopsies for Cancer: Coming to a Patient near You

by Nithya Krishnamurthy, Emily Spencer, Ali Torkamani and Laura Nicholson

J. Clin. Med. 2017, 6(1), 3; https://doi.org/10.3390/jcm6010003 - 4 Jan 2017

Cited by 71 | Viewed by 12919

Abstract

The use of circulating tumor DNA (ctDNA) as a novel and non-invasive test for the diagnosis and surveillance of cancer is a rapidly growing area of interest, with sequencing of ctDNA acting as a potential surrogate for tissue biopsy. Circulating tumor DNA has [...] Read more.

The use of circulating tumor DNA (ctDNA) as a novel and non-invasive test for the diagnosis and surveillance of cancer is a rapidly growing area of interest, with sequencing of ctDNA acting as a potential surrogate for tissue biopsy. Circulating tumor DNA has been detected incidentally during noninvasive prenatal testing and additionally in more than 75% of known cancer patients participating in ctDNA studies evaluating its sensitivity. In the setting of mutation-based targeted tumor therapy, it shows a concordance rate >80% when compared with gold-standard tissue biopsies. Through ctDNA detection and sequencing, a simple blood test becomes a liquid biopsy for cancer, surveying a patient’s entire circulation with the goal of early detection, prognostic information, personalized therapy options, and tracking for recurrence or resistance, all with fewer or no tissue biopsies. Given the recent first-ever FDA approval of a liquid biopsy, it is important for clinicians to be aware of the rapid advancements likely to bring these tests into our practices soon. Here we review the biology, clinical implications, and recent advances in circulating tumor DNA analysis. Full article

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Open AccessReview

A Systematic Review of the Role of Dysfunctional Wound Healing in the Pathogenesis and Treatment of Idiopathic Pulmonary Fibrosis

by Alan Betensley, Rabab Sharif and Dimitrios Karamichos

J. Clin. Med. 2017, 6(1), 2; https://doi.org/10.3390/jcm6010002 - 26 Dec 2016

Cited by 86 | Viewed by 9032

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disorder showcasing an interaction between genetic predisposition and environmental risks. This usually involves the coaction of a mixture of cell types associated with abnormal wound healing, leading to structural distortion and loss of [...] Read more.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disorder showcasing an interaction between genetic predisposition and environmental risks. This usually involves the coaction of a mixture of cell types associated with abnormal wound healing, leading to structural distortion and loss of gas exchange function. IPF bears fatal prognosis due to respiratory failure, revealing a median survival of approximately 2 to 3 years. This review showcases the ongoing progress in understanding the complex pathophysiology of IPF and it highlights the latest potential clinical treatments. In IPF, various components of the immune system, particularly clotting cascade and shortened telomeres, are highly involved in disease pathobiology and progression. This review also illustrates two US Food and Drug Administration (FDA)-approved drugs, nintedanib (OFEV, Boehringer Ingelheim, Ingelheim am Rhein, Germany) and pirfenidone (Esbriet, Roche, Basel, Switzerland), that slow IPF progression, but unfortunately neither drug can reverse the course of the disease. Although the mechanisms underlying IPF remain poorly understood, this review unveils the past and current advances that encourage the detection of new IPF pathogenic pathways and the development of effective treatment methods for the near future. Full article

(This article belongs to the Special Issue Chronic Respiratory Diseases)

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Open AccessArticle

The Effects of Ascorbate, N-Acetylcysteine, and Resveratrol on Fibroblasts from Patients with Mitochondrial Disorders

by Liza Douiev, Devorah Soiferman, Corinne Alban and Ann Saada

J. Clin. Med. 2017, 6(1), 1; https://doi.org/10.3390/jcm6010001 - 22 Dec 2016

Cited by 27 | Viewed by 8346

Abstract

Reactive oxygen species (ROS) are assumed to be implicated in the pathogenesis of inborn mitochondrial diseases affecting oxidative phosphorylation (OXPHOS). In the current study, we characterized the effects of three small molecules with antioxidant properties (N-acetylcysteine, ascorbate, and resveratrol) on ROS [...] Read more.

Reactive oxygen species (ROS) are assumed to be implicated in the pathogenesis of inborn mitochondrial diseases affecting oxidative phosphorylation (OXPHOS). In the current study, we characterized the effects of three small molecules with antioxidant properties (N-acetylcysteine, ascorbate, and resveratrol) on ROS production and several OXPHOS parameters (growth in glucose free medium, ATP production, mitochondrial content and membrane potential (MMP)), in primary fibroblasts derived from seven patients with different molecularly defined and undefined mitochondrial diseases. N-acetylcysteine appeared to be the most beneficial compound, reducing ROS while increasing growth and ATP production in some patients’ cells. Ascorbate showed a variable positive or negative effect on ROS, ATP production, and mitochondrial content, while incubation with resveratrol disclosed either no effect or detrimental effect on ATP production and MMP in some cells. The individual responses highlight the importance of investigating multiple parameters in addition to ROS to obtain a more balanced view of the overall effect on OXPHOS when evaluating antioxidant treatment options for mitochondrial diseases. Full article

(This article belongs to the Special Issue The Role of Oxidant Stress in Disease)

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Figure 1
Reactive oxygen species (ROS) production. Fibroblasts in microtiter wells were incubated in the absence or presence of the compounds for 72 h, ROS production (relative fluorescence units, RFU) was determined and normalized to cell content (A620). Results are presented as mean ± SEM of triplicate wells of at least two independent experiments. * p < 0.05 compared to mean of 3 controls in the corresponding medium, $ p < 0.05 compared to individual patient cells without additive. Full article ">Figure 2
Growth in glucose-free medium. Equal numbers of fibroblasts were seeded in microtiter wells in glucose-free (GAL) medium in the absence or presence of the compounds for 72 h. Growth was determined by measuring cell content by methylene blue (A620). Results are presented as mean ± SEM of triplicate wells of at least two independent experiments. * p < 0.05 compared to mean of 3 controls in the corresponding medium, $ p < 0.05 compared to individual patient cells without additive. Full article ">Figure 3
ATP production. Cells were grown in the absence or presence of the compounds for 72 h in microtiter wells. Subsequently, wells were washed, permeabilized, and incubated with glutamate and malate in the presence of ADP; ATP produced was analyzed by luciferin–luciferase (relative luminescence units, RLU). Results were normalized to cell content measured in parallel wells (A620). Results are presented as mean ± SEM of triplicate wells of at least two independent experiments. * p < 0.05 compared to mean of 3 controls in the corresponding medium, $ p < 0.05 compared to individual patient cells without additive. Full article ">Figure 4
Mitochondrial content. Cells were grown in the absence or presence of the compounds for 72 h in microtiter wells, and stained live with MitoTracker Green (RFU). Results were normalized to cell content measured in parallel wells (A620). Results are presented as mean ± SEM of triplicate wells of at least two independent experiments. * p < 0.05 compared to mean of 3 controls in the corresponding medium, $ p < 0.05 compared to individual patient cells without additive. Full article ">Figure 5
Mitochondrial membrane potential (MMP). Cells were grown in the absence or presence of the compounds for 72 h in microtiter wells, and stained live with tetramethylrhodamine ethyl ester (TMRE) (RED) and MitoTracker Green (GREEN). Results are presented as RED/GREEN ratio mean ± SEM of triplicate wells of at least two independent experiments. * p < 0.05 compared to mean of 3 controls in the corresponding medium, $ p < 0.05 compared to individual patient cells without additive. Full article ">

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J. Clin. Med., Volume 6, Issue 1 (January 2017) – 12 articles

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