Possible Role of IL-6R/STAT3/MiRNA-34a Feedback Loop in Osteosarcoma.

OBJECTIVE: Osteosarcoma is considered the most common primary malignant tumor that develops from the primary osteoblasts. MiRNAs are small non-coding RNAs that play a key role in tumorigenesis. The aim of this study was to detect the possible relationship between expression levels of miRNA-34a and levels of Signal transducer and activator of transcription 3 (STAT3) and interleukin-6 receptor (IL-6R) in osteosarcoma and the possible role of this relationship in development of metastases in these patients. METHODS: A total of thirty-six (36) bone samples were included in the study. They were divided into 3 groups: Group (I): Twelve normal bone samples as control group. Group (II): Twelve patients with non-metastatic osteosarcoma. Group (III): Twelve patients with metastatic osteosarcoma. MiRNA-34a expression levels were estimated using qRT-PCR. STAT3 and IL-6R levels were measured by ELISA. RESULTS: Expression level of miRNA-34a was downregulated in osteosarcoma groups compared to control group. STAT3 and IL-6R levels were upregulated in osteosarcoma groups compared to control group. This difference in expression levels was found to be more significant in the metastatic group than the non-metastatic one (P<0.001 each). There was a significant positive correlation between STAT3 and IL-6R (r=0.868, P<0.001), and a significant inverse correlation between IL6 and miRNA-34a (r=-0.993, P<0.001). CONCLUSION: miRNA-34a, STAT3 and IL-6R feedback loop could be a potential target for treatment of osteosarcoma and can be used as prognostic indicator for this disease.

Focused extracorporeal shockwave therapy for youth sports-related apophyseal injuries: case series.

BACKGROUND: Apophyseal overuse injuries are self-limited with skeletal maturity; however, they may be a source of significant pain and time lost from training. There is a lack of consensus for its management with the current available treatment, which might lag behind the ongoing development of regenerative medicine. The current retrospective case study aimed to assess the potential effectiveness and short-term safety of extracorporeal shockwave therapy (ESWT) in apophyseal injuries. METHODS: Data from 22 growing athletes [15 patients with Osgood-Schlatter disease and seven patients with Sever's disease] who received ESWT at a sports medicine unit in a university hospital were reviewed. All patients received low energy (= 0.1 mJ/mm2) level-focused ESWT using electrohydraulic generation technology. The clinical focusing technique was used upon applying ESWT. RESULTS: The number of sessions received to achieve full recovery ranged from 1 to 3 sessions. The time from treatment initiation to previous activity level was 2 weeks in 14 patients (63.3%), 4 weeks in seven patients (31.8%) and 11 weeks in one patient (4.5%). No adverse events were reported. No recurrence occurred up to 3 months after the last session. CONCLUSION: ESWT is a potentially safe and effective treatment for apophyseal injuries. It may facilitate an early return to sport activities.

Vitamin D and miRNA-155 in Behçet's Disease: Possible Association with the Disease and Disease Activity.

Background: Vitamin D (vit D) controls inflammation and immunity. In Behçet's disease (BD), microRNA-155 is recognized as a significant immune response regulator. We aimed to investigate the role of vit D on immunomodulation and downregulation of inflammatory pathways associated with BD and detect the role of miRNA-155 in BD. Methods: miRNA-155 expression by Real Time -Polymerase Chain Reaction (RT-PCR), and vit D, nuclear factor Kappa-light-chain-enhancer of activated B cells (NF-κB), and Tumor necrosis fact of TNF-α) expression by Enzyme Linked Immunosorbent Assay (ELISA) were assessed. Results: BD patients had a significantly higher relative expression of microRNA-155 (P< 0.001), it was significantly related to vascular manifestations (P< 0.001). Vit D relative expression was significantly low in BD (P< 0.001). There was a significant rise in miRNA-155 in the active group compared to the inactive group (P< 0.001). A significant decrease in vit D levels (IU) was found in inactive and active individuals suffering from BD when compared to controls (P< 0.001). A significant rise was found in vit D levels in inactive BD cases (P< 0.001). A significant positive correlations were found between miRNA-155, NF-κB, TNF-α, and negative correlations with vit D relative expression in BD patients. Conclusions: miRNA-155 relative expression is higher in BD is significantly related to vascular manifestations. It may have a relationship to disease activity. Vitamin D relative expression is significantly low in BD patients, which can significantly influence immunomodulatory BD therapy. Vitamin D deficiency linked to active BD.

Downregulation of aquaporin 3 in patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease. Aquaporin 3 (AQP3) is a glycerol/water channel involved in several physiological functions. Evaluation of the tissue expression and localization of AQP3 in the skin of PV patients. Twenty-seven PV patients and 30 controls were included. The patients were subjected to history taking, clinical evaluation, Autoimmune Bullous Skin Disorder Intensity Score and 4-mm punch biopsy. The biopsies were stained using anti-human AQP3 antibody and the immunofluorescence pattern and intensity were evaluated using a scoring system and ImageJ software analysis. AQP3 was expressed in the basal epidermis in 27 (100%) and in the suprabasal epidermis in 19 PV patients (70.4%). It was expressed in all controls in basal and suprabasal layers. Intensity of AQP3 immunofluorescence was strong in 2 (7.4%), moderate in 19 (70.4%) and weak in 6 patients (22.2%) while it was strong in 18 (60%) and moderate in 12 controls (40%). AQP3 expression was significantly lower in patients than controls in the suprabasal epidermis (p = 0.001). Patients with extensive disease had significantly weaker AQP3 intensity than those with marked disease (p = 0.005) Downregulation of AQP3 in patients with PV, especially in the suprabasal layers and in extensive clinical disease, suggests a potential role of AQP3 in the pathogenesis of PV.

Dermoscopic evaluation of tranexamic acid versus Vitamin C, with microneedling in the treatment of melasma: a comparative, split-face, single-blinded study.

BACKGROUND: Melasma is a complex pigmentary disorder with challenging management. OBJECTIVES: Evaluation of efficacy of topical tranexamic acid (TXA) versus Vitamin C (Vit C) with microneedling (MN) in melasma therapy. MATERIALS AND METHODS: In 30 females with melasma, after 4 weeks of using Modified Kligman's formula, the right side of the face was treated with MN + TXA and the left with MN + Vit C for five biweekly sessions. Wood's light, dermoscopy, Melasma Area and Severity Index (MASI), MASI malar right and malar left (MASIMR and MASIML), Visual Analogue Score (VAS), and Dermatology Life Quality Index (DLQI) were evaluated at weeks 0, 4, 12, and 16. RESULTS: Both MASIMR and MASIML decreased significantly (p < .001). Both sides exhibited significant diminution in dark fine granules (p-value < .001), homogeneous pigmentation (p-value = .005) and pseudoreticular brown network (p-value = .028). However, telangiectasia significantly improved only on the TXA treated side (p = .002). DLQI improved significantly on both sides (p < .001). In some patients transformation of mixed to dermal melasma was depicted. CONCLUSION: MN followed by Vit C or TXA is successful in melasma management, the latter being advantageous vis-a-vis dermal vascularity and epidermal pigmentation. Dermoscopic evaluation appears crucial in choosing optimum treatment in each patient.

Downregulation of interleukin 36γ and its cleaver cathepsin G following treatment with narrow-band ultraviolet B phototherapy in psoriasis vulgaris.

BACKGROUND: Growing evidence suggests the important role of IL-36 in the pathogenesis of psoriasis. Cathepsin G is a neutrophil-derived protease that can activate IL-36γ. OBJECTIVE: To assess the expression of IL-36γ and cathepsin G in psoriasis and to quantify the impact of treatment with narrow-band ultraviolet B phototherapy (NB-UVB) on their levels. METHODS: This case-control study involved 26 patients with moderate-severe psoriasis and 25 healthy volunteers. Psoriasis patients eligible for phototherapy received 24 NB-UVB sessions. Punch skin biopsies were obtained from all participants at recruitment and after phototherapy from patients. Real-time PCR was utilized for quantitative assessment of IL-36γ and cathepsin G expression in tissue samples. RESULTS: The expression of IL-36γ and cathepsin G was significantly higher in psoriasis before NB-UVB therapy compared to controls (p < .001). Both proteins decreased significantly with clinical improvement following NB-UVB therapy compared to baseline (p < .001). However, their expression after treatment was still higher than controls (p < .001). CONCLUSION: IL-36γ and cathepsin G expression is upregulated in psoriatic lesions, supporting their role as mediators of inflammation in psoriasis. Downregulation of IL-36γ and cathepsin G is a possible mechanism for psoriasis improvement after NB-UVB therapy. IL-36 and cathepsin G can be considered as therapeutic targets for psoriasis.

Trichoscopic features of hair and scalp in noncomplaining individuals: A descriptive study.

BACKGROUND: Trichoscopy is a simple noninvasive tool that is used in calculating different hair parameters and the diagnosis of different hair/scalp diseases at variable magnifications. OBJECTIVE: The aim of this work is to describe the features and parameters of hair and scalp in a healthy sample of Egyptian population using videodermoscopy. This may help to provide standard range of measurements of normal hair in both males and females. METHODS: A nonrandomized, observational study for features and parameters of hair structure, performed on 368 healthy Egyptian subjects with no hair/scalp complaint. RESULTS: Each scalp area has its own vascular pattern. Male subjects showed different values regarding their hair parameters from the female subjects. No significant difference was seen after application of hair dye, except for few values. In both genders, there was a change in hair parameters with age. Smoking had a negative influence on hair parameters. CONCLUSION: Hair features and parameters observed in our population are different from those reported by other populations. Smoking affects hair thickness and density. Dyed hair has greater thickness mostly due to the protective effect of hair conditioners.

Protective effect of cerium oxide nanoparticles on cisplatin and oxaliplatin primary toxicities in male albino rats.

Cisplatin and oxaliplatin are widely used anticancer drugs. Their use is restricted by their dose-limiting side effects: nephrotoxicity and neurotoxicity, respectively. Cerium oxide nanoparticles (CONPs) are promising antioxidant and anti-inflammatory agent. To test the possible ameliorative impact of CONPs on the toxic effect of cisplatin and oxaliplatin in male albino rats. Forty eight rats were divided into 6 groups: control group, CONPs group, cisplatin group, cisplatin and CONPs group, oxaliplatin group, and oxaliplatin and CONPs group. After 4 weeks, serum urea and creatinine, renal tissue level of interleukin 10 (IL10), and total antioxidant (TAO) were measured in control, CONPs, and cisplatin groups. The other kidney was used for histopathological and immunohistochemical studies. The right sciatic nerves and the lumbar spinal cord of rats from control, CONPs, and oxaliplatin groups were used for immunohistochemical evaluations of nitrotyrosine, myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP). Cisplatin significantly increased serum urea and creatinine levels, significantly decreased the kidney level of IL10 and TAO with marked tubular necrosis, hemorrhage and renal damage. Also, it decreased IL10 immunohistochemical expression. CONPs significantly decreased the serum urea and creatinine level and increased IL10 and TAO with lower renal damage and strong IL10 expression compared with cisplatin group. Oxaliplatin significantly decreased MBP immunoreactivity and increased nitrotyrosine immunoreactivity. In the lumbar spinal cord, GFAP immunoreactivity was significantly increased. CONPs significantly increased MBP and decreased nitrotyrosine immunoreactivity. GFAP immunoreactivity was significantly decreased. CONPs ameliorated cisplatin and oxaliplatin primary toxicities through anti-inflammatory and antioxidant characteristics.

Downregulation of aquaporin 3 in bullous pemphigoid patients.

Bullous pemphigoid (BP) is a chronic autoimmune skin disease. Aquaporin 3 (AQP 3) has a possible role in the pathogenesis of many dermatological diseases. In this work, we aimed to evaluate the expression of AQP 3 in BP. Perilesional skin biopsies were taken from 24 BP patients and 13 controls. The biopsies were stained by direct immunofluorescence using rabbit anti-human AQP 3 FITC antibody. The expression of AQP 3 was weak in 5 patients (20.8%), moderate in 18 patients (75%), strong in 1 patient (4.2%) in the suprabasal layers. It was negative in 4 patients (16.7%), weak in 18 patients (75%), moderate in 2 patients (8.3%) and no strong fluorescence was seen in the basal layers. In the controls, the expression was strong in ten controls (76.9%), moderate in three controls (23.1%) and no controls showed weak fluorescence in the suprabasal layer. The basal layer showed strong fluorescence in 11 controls (84.6%), moderate in 2 controls (15.4%) and no controls showed mild or no fluorescence. There was a statistically significant difference in the expression of AQP 3 between basal and suprabasal layers of BP patients but not of the controls. There was statistically significant difference in the expression of AQP 3 between patients and controls in both the basal (P value < 0.001) and the suprabasal layers (P value < 0.001). In conclusion, AQP 3 was downregulated in BP patients especially in the basal cell layer. This suggests that AQP 3 plays a role in the pathogenesis of BP.

Role of adipose tissue derived stem cells differentiated into insulin producing cells in the treatment of type I diabetes mellitus.

Generation of new ß cells is an important approach in the treatment of type 1 diabetes mellitus (type 1 DM). Adipose tissue-derived stem cells (ADSCs) might be one of the best sources for cell replacement therapy for diabetes. Therefore, this work aimed to test the possible role of transplanted insulin-producing cells (IPCs) differentiated from ADSCs in treatment of streptozotocin (STZ) induced type I DM in rats. Type 1 DM was induced by single intra peritoneal injection with STZ (50 mg/kg BW). Half of the diabetic rats were left without treatment and the other half were injected with differentiated IPCs directly into the pancreas. ADSCs were harvested, cultured and identified by testing their phenotypes through flow cytometry. They were further subjected to differentiation into IPCs using differentiation medium. mRNA expression of pancreatic transcription factors (pdx1), insulin and glucose transporter-2 genes by real time PCR was done to detect the cellular differentiation and confirmed by stimulated insulin secretion. The pancreatic tissues from all groups were examined 2 months after IPC transplantation and were subjected to histological, Immunohistochemical and morphometric study. The differentiated IPCs showed significant expression of pancreatic ß cell markers and insulin secretion in glucose dependent manner. Treatment with IPCs induced apparent regeneration, diffused proliferated islet cells and significant increase in C-peptide immune reaction. We concluded that transplantation of differentiated IPCs improved function and morphology of Islet cells in diabetic rats. Consequently, this therapy option may be a promising therapeutic approach to patient with type 1 DM if proven to be effective and safe.

Cancer cell-soluble factors reprogram mesenchymal stromal cells to slow cycling, chemoresistant cells with a more stem-like state.

BACKGROUND: Mesenchymal stem cells (MSCs) play different roles in modulating tumor progression, growth, and metastasis. MSCs are recruited to the tumor site in large numbers and subsequently have an important microenvironmental role in modulating tumor progression and drug sensitivity. However, the effect of the tumor microenvironment on MSC plasticity remains poorly understood. Herein, we report a paracrine effect of cancer cells, in which they secrete soluble factors that promote a more stem-like state in bone marrow mesenchymal stem cells (BM-MSCs). METHODS: The effect of soluble factors secreted from MCF7, Hela, and HepG2 cancer cell lines on BM-MSCs was assessed using a Transwell indirect coculture system. After 5 days of coculture, BM-MSCs were characterized by flow cytometry for surface marker expression, by qPCR for gene expression profile, and by confocal immunofluorescence for marker expression. We then measured the sensitivity of cocultured BM-MSCs to chemotherapeutic agents, their cell cycle profile, and their response to DNA damage. The sphere formation, invasive properties, and in-vivo performance of BM-MSCs after coculture with cancer cells were also measured. RESULTS: Indirect coculture of cancer cells and BM-MSCs, without direct cell contact, generated slow cycling, chemoresistant spheroid stem cells that highly expressed markers of pluripotency, cancer cells, and cancer stem cells (CSCs). They also displayed properties of a side population and enhanced sphere formation in culture. Accordingly, these cells were termed cancer-induced stem cells (CiSCs). CiSCs showed a more mesenchymal phenotype that was further augmented upon TGF-ß stimulation and demonstrated a high expression of the ß-catenin pathway and ALDH1A1. CONCLUSIONS: These findings demonstrate that MSCs, recruited to the tumor microenvironment in large numbers, may display cellular plasticity, acquire a more stem-like state, and acquire some properties of CSCs upon exposure to cancer cell-secreted factors. These acquired characteristics may contribute to tumor progression, survival, and metastasis. Our findings provide new insights into the interactions between MSCs and cancer cells, with the potential to identify novel molecular targets for cancer therapy.

Adipose Stem Cells Display Higher Regenerative Capacities and More Adaptable Electro-Kinetic Properties Compared to Bone Marrow-Derived Mesenchymal Stromal Cells.

Adipose stem cells (ASCs) have recently emerged as a more viable source for clinical applications, compared to bone-marrow mesenchymal stromal cells (BM-MSCs) because of their abundance and easy access. In this study we evaluated the regenerative potency of ASCs compared to BM-MSCs. Furthermore, we compared the dielectric and electro-kinetic properties of both types of cells using a novel Dielectrophoresis (DEP) microfluidic platform based on a printed circuit board (PCB) technology. Our data show that ASCs were more effective than BM-MSCs in promoting neovascularization in an animal model of hind-limb ischemia. When compared to BM-MSCs, ASCs displayed higher resistance to hypoxia-induced apoptosis, and to oxidative stress-induced senescence, and showed more potent proangiogenic activity. mRNA expression analysis showed that ASCs had a higher expression of Oct4 and VEGF than BM-MSCs. Furthermore, ASCs showed a remarkably higher telomerase activity. Analysis of the electro-kinetic properties showed that ASCs displayed different traveling wave velocity and rotational speed compared to BM-MSCs. Interestingly, ASCs seem to develop an adaptive response when exposed to repeated electric field stimulation. These data provide new insights into the physiology of ASCs, and evidence to their potential superior potency compared to marrow MSCs as a source of stem cells.

In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide interventions.

Hsa-miR-195 targets PCMT1 in hepatocellular carcinoma that increases tumor life span.

MicroRNAs are small 19-25 nucleotides which have been shown to play important roles in the regulation of gene expression in many organisms. Downregulation or accumulation of miRNAs implies either tumor suppression or oncogenic activation. In this study, differentially expressed hsa-miR-195 in hepatocellular carcinoma (HCC) was identified and analyzed. The prediction was done using a consensus approach of tools. The validation steps were done at two different levels in silico and in vitro. FGF7, GHR, PCMT1, CITED2, PEX5, PEX13, NOVA1, AXIN2, and TSPYL2 were detected with high significant (P < 0.005). These genes are involved in important pathways in cancer like MAPK signaling pathway, Jak-STAT signaling pathways, regulation of actin cytoskeleton, angiogenesis, Wnt signaling pathway, and TGF-beta signaling pathway. In vitro target validation was done for protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1). The co-transfection of pmirGLO-PCMT1 and pEGP-miR-195 showed highly significant results. Firefly luciferase was detected using Lumiscensor and t test analysis was done. Firefly luciferase expression was significantly decreased (P < 0.001) in comparison to the control. The low expression of firefly luciferase validates the method of target prediction that we used in this work by working on PCMT1 as a target for miR-195. Furthermore, the rest of the predicted genes are suspected to be real targets for hsa-miR-195. These target genes control almost all the hallmarks of liver cancer which can be used as therapeutic targets in cancer treatment.

An integrative meta-analysis of microRNAs in hepatocellular carcinoma.

We aimed to shed new light on the roles of microRNAs (miRNAs) in liver cancer using an integrative in silico bioinformatics analysis. A new protocol for target prediction and functional analysis is presented and applied to the 26 highly differentially deregulated miRNAs in hepatocellular carcinoma. This framework comprises: (1) the overlap of prediction results by four out of five target prediction tools, including TargetScan, PicTar, miRanda, DIANA-microT and miRDB (combining machine-learning, alignment, interaction energy and statistical tests in order to minimize false positives), (2) evidence from previous microarray analysis on the expression of these targets, (3) gene ontology (GO) and pathway enrichment analysis of the miRNA targets and their pathways and (4) linking these results to oncogenesis and cancer hallmarks. This yielded new insights into the roles of miRNAs in cancer hallmarks. Here we presented several key targets and hundreds of new targets that are significantly enriched in many new cancer-related hallmarks. In addition, we also revealed some known and new oncogenic pathways for liver cancer. These included the famous MAPK, TGFß and cell cycle pathways. New insights were also provided into Wnt signaling, prostate cancer, axon guidance and oocyte meiosis pathways. These signaling and developmental pathways crosstalk to regulate stem cell transformation and implicate a role of miRNAs in hepatic stem cell deregulation and cancer development. By analyzing their complete interactome, we proposed new categorization for some of these miRNAs as either tumor-suppressors or oncomiRs with dual roles. Therefore some of these miRNAs may be addressed as therapeutic targets or used as therapeutic agents. Such dual roles thus expand the view of miRNAs as active maintainers of cellular homeostasis.