Changes in aminoacidergic and monoaminergic neurotransmission in the hippocampus and amygdala of rats after ayahuasca ingestion

AIM: To evaluate changes in neurotransmission induced by a psychoactive beverage ayahuasca in the hippocampus and amygdala of naive rats. METHODS: The level of monoamines, their main metabolites and amino acid neurotransmitters concentrations were quantified using high performance liquid chromatography(HPLC). Four groups of rats were employed: saline-treated and rats receiving 250, 500 and 800 mg/kg of ayahuasca infusion(gavage). Animals were killed 40 min after drug ingestion and the structures stored at-80 ℃ until HPLC assay. The data from all groups were compared using Analysis of variance and Scheffé as post test and P 0.05 was accepted as significant. RESULTS: The results showed decreased concentrations of glycine(GLY)(0.13 ± 0.03 vs 0.29 ± 0.07, P 0.001) and γ-aminobutyric acid(GABA)(1.07 ± 0.14 vs 1.73 ± 0.25, P 0.001) in the amygdala of rats that received 500 of ayahuasca. Animals that ingested 800 mg/kg of ayahuasca also showed a reduction of GLY level(0.11 ± 0.01 vs 0.29 ± 0.07, P 0.001) and GABA(0.98 ± 0.06 vs 1.73 ± 0.25, P 0.001). In the hippocampus, increased GABA levels were found in rats that received all ayahuasca doses: 250 mg/kg(1.29 ± 0.19 vs 0.84 ± 0.21, P 0.05); 500 mg/kg(2.23 ± 038 vs 084 ± 0.21, P 0.05) and 800 mg/kg(1.98 ± 0.92 vs 0.84 ± 0.21, P 0.05). In addition, an increased utilization rate of all monoamines was found in the amygdala after ayahuasca administration in doses: 250 mg/kg(noradrenaline: 0.16 ± 0.02 vs 0.36 ± 0.06, P 0.01; dopamine: 0.39 ± 0.012 vs 2.39 ± 0.84, P 0.001; serotonin: 1.02 ± 0.22 vs 4.04 ± 0.91, P 0.001), 500 mg/kg(noradrenaline: 0.08 ± 0.02 vs 0.36 ± 0.06, P 0.001; dopamine: 0.33 ± 0.19 vs 2.39 ± 0.84, P 0.001; serotonin: 0.59 ± 0.08 vs 4.04 ± 0.91, P 0.001) and 800 mg/kg(noradrenaline: 0.16 ± 0.04 vs 0.36 ± 0.06, P 0.001; dopamine: 0.84 ± 0.65 vs2.39 ± 0.84, P 0.05; serotonin: 0.36 ± 0.02 vs 4.04 ± 0.91, P 0.001). CONCLUSION: Our data suggest increased release of inhibitory amino acids by the hippocampus and an increased utilization rate of monoamines by the amygdala after different doses of ayahuasca ingestion.     

Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells

AIM: To explore the approaches exerted by mesenchymal stem cells(MSCs) to improve Parkinson's disease(PD) pathophysiology.METHODS: MSCs were harvested from bone marrowof femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group(1) was control, Groups(2) and(3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group(2) was left untreated, while Group(3) was treated with single intravenous dose of bone marrow derived MSCs(BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1(TGF-β1), monocyte chemoattractant protein-1(MCP-1) and brain derived neurotrophic factor(BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase(TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done.RESULTS: BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1(489.7 ± 13.0 vs 691.2 ± 8.0, P 0.05) and MCP-1(89.6 ± 2.0 vs 112.1 ± 1.9, P 0.05) levels associated with significant increase in serum BDNF(3663 ± 17.8 vs 2905 ± 72.9, P 0.05) and brain DA(874 ± 15.0 vs 599 ± 9.8, P 0.05) levels as well as brain TH(1.18 ± 0.004 vs 0.54 ± 0.009, P 0.05) and nestin(1.29 ± 0.005 vs 0.67 ± 0.006, P 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin(293.2 ± 15.9 vs 271.5 ± 15.9, P 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs. CONCLUSION: The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.     

Protein Kinase G1 α Overexpression Increases Stem Cell Survival and Cardiac Function after Myocardial Infarction

Background

We hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α) could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs) contributing to regeneration of the ischemic heart.

Methods and Results

MSCs from male rats were transduced with adenoviral vector encoding for PKG1α (^PKG1αMSCs).Controls included native MSCs (^NatMSCs) and MSCs transduced with an empty vector (^NullMSCs). PKG1α activity was increased approximately 20, 5 and 16 fold respectively in ^PKG1αMSCs. ^PKG1αMSCs showed improved survival under oxygen and glucose deprivation (OGD) which was evidenced by lower LDH release, caspase-3/7 activity and number of positive TUNEL cells. Anti-apoptotic proteins pAkt, pGSK3β, and Bcl-2 were significantly increased in ^PKG1αMSCs compared to ^NatMSCs and ^NullMSCs. Higher release of multiple prosurvival and angiogenic factors such as HGF, bFGF, SDF-1 and Ang-1 was observed in ^PKG1αMSCs before and after OGD. In a female rat model of acute myocardial infarction, ^PKG1αMSCs group showed higher survival compared with ^NullMSCs group at 3 and 7 days after transplantation as determined by TUNEL staining and sry-gene quantitation by real-time PCR. Increased anti-apoptotic proteins and paracrine factors in vitro were also identified. Immunostaining for cardiac troponin I combined with GFP showed increased myogenic differentiation of ^PKG1αMSCs. At 4 weeks after transplantation, compared to DMEM group and ^NullMSCs group, ^PKG1αMSCs group showed increased blood vessel density in infarct and peri-infarct areas (62.5±7.7; 68.8±7.3 per microscopic view, p<0.05) and attenuated infarct size (27.2±2.5%, p<0.01). Heart function indices including ejection fraction (52.1±2.2%, p<0.01) and fractional shortening (24.8%±1.3%, p<0.01) were improved significantly in ^PKG1αMSCs group.

Conclusion

Overexpression of PKG1α transgene could be a powerful approach to improve MSCs survival and their angiomyogenic potential in the infarcted heart.     

Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis

AIM: To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis.METHODS: The fKSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3^rd passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of fKSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of fKSC in ARF, fKSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nick-end labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α and endothelial nitric oxide synthase (eNOS) by western blot.RESULTS: The fKSC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well as renal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin (CK)18 and CK19 positive epithelial cells. Administration of fKSC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3 (2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7 (0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fKSC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis (P < 0.05) of renal cells. The kidneys of fKSC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields (HPF), P < 0.05], and on day 7 (21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fKSC treated rats had an up-regulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eNOS on day 3 (P < 0.05).CONCLUSION: Our study shows that fKSC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.     

Adult patients with congenital adrenal hyperplasia have elevated blood pressure but otherwise a normal cardiovascular risk profile

Objective

Treatment with glucocorticoids and mineralocorticoids has changed congenital adrenal hyperplasia (CAH) from a fatal to a chronic lifelong disease. Long-term treatment, in particular the chronic (over-)treatment with glucocorticoids, may have an adverse effect on the cardiovascular risk profile in adult CAH patients. The objective of this study was to evaluate the cardiovascular risk profile of adult CAH patients.

Design

Case-control study.

Patients and Measurements

In this case-control study the cardiovascular risk profile of 27 adult CAH patients and 27 controls, matched for age, sex and body mass index was evaluated by measuring ambulatory 24-hour blood pressure, insulin sensitivity (HOMA-IR), lipid profiles, albuminuria and circulating cardiovascular risk markers (PAI-1, tPA, uPA, tPA/PAI-1 complex, hsCRP, adiponectin, IL-6, IL-18 and leptin).

Results

24-Hour systolic (126.3 mmHg±15.5 vs 124.8 mmHg±15.1 in controls, P = 0.019) and diastolic (76.4 mmHg±12.7 vs 73.5 mmHg±12.4 in controls, P<0.001) blood pressure was significantly elevated in CAH patients compared to the control population. CAH patients had higher HDL cholesterol levels (P<0.01), lower hsCRP levels (P = 0.03) and there was a trend toward elevated adiponectin levels compared to controls. Other cardiovascular risk factors were similar in both groups.

Conclusion

Adult CAH patients have higher ambulatory blood pressure compared to healthy matched controls. Other cardiovascular risk markers did not differ, while HDL-cholesterol, hsCRP and adiponectin levels tended to be more favorable.     

Moderate- and high-intensity exhaustive exercise in the heat induce a similar increase in monocyte Hsp72

This study examined the relationship between exhaustive exercise in the heat at moderate and high intensities on the intracellular heat shock protein 72 (iHsp72) response. Twelve male subjects cycled to exhaustion at 60 and 75 % of maximal oxygen uptake in hot conditions (40 °C, 50 % RH). iHsp72 concentration was measured in monocytes before, at exhaustion and 24 h after exercise. Rectal temperature, heart rate and oxygen uptake were recorded during exercise. Volitional exhaustion occurred at 58.9 ± 12.1 and 27.3 ± 9.5 min (P < 0.001) and a rectal temperature of 39.8 ± 0.4 and 39.2 ± 0.6 °C (P = 0.002), respectively, for 60 and 75 %. The area under the curve above a rectal temperature of 38.5 °C was greater at 60 % (17.5 ± 6.6 °C min) than 75 % (3.4 ± 4.8 °C min; P < 0.001), whereas the rate of increase in rectal temperature was greater at 75 % (5.1 ± 1.7 vs. 2.2 ± 1.4 °C h⁻¹; P < 0.001). iHsp72 concentration increased similarly at exhaustion relative to pre-exercise (P = 0.044) and then increased further at 24 h (P < 0.001). Multiple regression analysis revealed no predictor variables associated with iHsp72 expression; however, a correlation was observed between exercise intensities for the increase in iHsp expression at exhaustion and 24 h (P < 0.05). These results suggest that iHsp72 expression increased in relation to the level of hyperthermia attained and sustained at 60 % and the higher metabolic rate and greater rate of increase in core temperature at 75 %, with the further increase in iHsp72 concentration 24 h after exercise reinforcing its role as a chaperone and cytoprotective agent.     

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm³ after 3 days, 1.24 ± 0.76 mm³ after 7 days, 0.33 ± 0.03 mm³ after 14 days, and 0.09 ± 0.05 mm³ after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.     

Metabolic Consequences of High-Fat Diet Are Attenuated by Suppression of HIF-1��

Obesity is associated with tissue hypoxia and the up-regulation of hypoxia inducible factor 1 alpha (HIF-1α). Prior studies in transgenic mice have shown that HIF-1α plays a role in the metabolic dysfunction associated with obesity. Therefore, we hypothesized that, after the development of diet-induced obesity (DIO), metabolic function could be improved by administration of HIF-1α antisense oligonucleotides (ASO). DIO mice were treated with HIF-1α ASO or with control ASO for 8 weeks and compared with an untreated group. We found that HIF-1α ASO markedly suppressed Hif-1α gene expression in adipose tissue and the liver. HIF-1α ASO administration induced weight loss. Final body weight was 41.6±1.4 g in the HIF-1α ASO group vs 46.7±0.9 g in the control ASO group and 47.9±0.8 g in untreated mice (p<0.001). HIF-1α ASO increased energy expenditure (13.3±0.6 vs 12±0.1 and 11.9±0.4 kcal/kg/hr, respectively, p<0.001) and decreased the respiratory exchange ratio (0.71±0.01 vs 0.75±0.01 and 0.76±0.01, respectively, p<0.001), which suggested switching metabolism to fat oxidation. In contrast, HIF-1a ASO had no effect on food intake or activity. HIF-1α ASO treatment decreased fasting blood glucose (195.5±8.4 mg/dl vs 239±7.8 mg/dl in the control ASO group and 222±8.2 mg/dl in untreated mice, p<0.01), plasma insulin, hepatic glucose output, and liver fat content. These findings demonstrate that the metabolic consequences of DIO are attenuated by HIF-1α ASO treatment.     

Muscle damage and its relationship with muscle fatigue during a half-iron triathlon

Background

To investigate the cause/s of muscle fatigue experienced during a half-iron distance triathlon.

Methodology/Principal Findings

We recruited 25 trained triathletes (36±7 yr; 75.1±9.8 kg) for the study. Before and just after the race, jump height and leg muscle power output were measured during a countermovement jump on a force platform to determine leg muscle fatigue. Body weight, handgrip maximal force and blood and urine samples were also obtained before and after the race. Blood myoglobin and creatine kinase concentrations were determined as markers of muscle damage.

Results

Jump height (from 30.3±5.0 to 23.4±6.4 cm; P<0.05) and leg power output (from 25.6±2.9 to 20.7±4.6 W · kg⁻¹; P<0.05) were significantly reduced after the race. However, handgrip maximal force was unaffected by the race (430±59 to 430±62 N). Mean dehydration after the race was 2.3±1.2% with high inter-individual variability in the responses. Blood myoglobin and creatine kinase concentration increased to 516±248 µg · L⁻¹ and 442±204 U · L⁻¹, respectively (P<0.05) after the race. Pre- to post-race jump change did not correlate with dehydration (r = 0.16; P>0.05) but significantly correlated with myoglobin concentration (r = 0.65; P<0.001) and creatine kinase concentration (r = 0.54; P<0.001).

Conclusions/significance

During a half-iron distance triathlon, the capacity of leg muscles to produce force was notably diminished while arm muscle force output remained unaffected. Leg muscle fatigue was correlated with blood markers of muscle damage suggesting that muscle breakdown is one of the most relevant sources of muscle fatigue during a triathlon.     

Efficient adenoviral-mediated gene delivery into porcine mesenchymal stem cells

Mesenchymal stem cell (MSC) mediated gene therapy research has been conducted predominantly on rodents. Appropriate large animal models may provide additional safety and efficacy information prior to human clinical trials. The objectives of this study were: (a) to optimize adenoviral transduction efficiency of porcine bone marrow MSCs using a commercial polyamine-based transfection reagent (GeneJammer, Stratagene, La Jolla, CA), and (b) to determine whether transduced MSCs retain the ability to differentiate into mesodermal lineages. Porcine MSCs (pMSCs) were infected under varying conditions, with replication-defective adenoviral vectors carrying the GFP gene and GFP expression analyzed. Transduced cells were induced to differentiate in vitro into adipogenic, chondrogenic, and osteogenic lineages. We observed a 5.5-fold increase in the percentage of GFP-expressing pMSCs when adenovirus type 5 carrying the adenovirus type 35 fiber (Ad5F35eGFP) was used in conjunction with GeneJammer. Transduction of pMSCs at 10.3-13.8 MOI (1,500-2,000 vp/cell) in the presence of Gene Jammer yielded the highest percentage of GFP-expressing cells ( approximately 90%) without affecting cell viability. A similar positive effect was detected when pMSCs were infected with an Ad5eGFP vector. Presence of fetal bovine serum (FBS) during adenoviral transduction enhanced vector-encoded transgene expression in both GeneJammer-treated and control groups. pMSCs transduced with adenovirus vector in the presence of GeneJammer underwent lipogenic, chondrogenic, and osteogenic differentiation. Addition of GeneJammer during adenoviral infection of pMSCs can revert the poor transduction efficiency of pMSCs while retaining their pluripotent differentiation capacity. GeneJammer-enhanced transduction will facilitate the use of adenoviral vectors in MSC-mediated gene therapy models and therapies.     

Novel links among peroxiredoxins,endothelial dysfunction,and severity of atherosclerosis in type 2 diabetic patients with peripheral atherosclerotic disease

Peroxiredoxins, a group of antioxidant protein enzymes (PRDX1 to 6), are reported as antiatherogenic factors in animals; however, human studies are lacking. The present work aims to provide baseline data regarding the phenotype of PRDX1, 2, 4, and 6 in diabetic patients with peripheral atherosclerosis disease (PAD) and their relation to endothelial dysfunction (ED) and disease severity. Plasma levels of PRDX1, 2, 4, and 6 and markers of endothelial dysfunction (ICAM-1 and VCAM-1) were measured using ELISA in 55 type 2 diabetic patients having PAD and 25 healthy subjects. Ankle–brachial index (ABI), body mass index (BMI), triglycerides (TG), total cholesterol, HbA1c, and insulin resistance (HOMA IR) were measured. PRDX1, 2, 4, and 6 levels were significantly higher in patients compared to controls (PRDX1 21.9 ± 5.71 vs 16.8 ± 3.9 ng/ml, P < 0.001, PRDX2 36.5 ± 14.83 vs 20.4 ± 8.61 ng/ml, P < 0.001, PRDX4 3,840 ± 1,440 vs 2,696 ± 1,972 pg/ml, P < 0.005, PRDX6 311 ± 110 vs 287.9 ± 114 pg/ml, P < 0.05). PRDX1 and PRDX4 correlated negatively with ABI (r = −0.273, P < 0.05 and r = −0.28, P < 0.05, respectively), while PRDX1 and PRDX2 correlated positively with HOMA/IR and TG (r = 0.276, P < 0.01 and r = 0.295, P < 0.01, respectively). ICAM-1 was associated with PRDX2 and log PRDX6 (r = 0.345, P = 0.0037 and r = 0.344, P = 0.0038). Our results provide strong links among PRDXs, ED, and severity of PAD in diabetic patients which warrants further evaluation to clarify whether high circulating levels of PRDXs are a consequence of chronic atherosclerotic disease or a predisposing factor for later cardiovascular events.     

Contribution of Baicalin on the Plasma Protein Binding Displacement and CYP3A Activity Inhibition to the Pharmacokinetic Changes of Nifedipine in Rats In Vivo and In Vitro

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C _max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC_0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), V_d increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C _max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism.     

Development and Clinical Evaluation of Clotrimazole–β-Cyclodextrin Eyedrops for the Treatment of Fungal Keratitis

Fungal keratitis is a serious corneal disease that may result in loss of vision. There are limited treatment options available in Iraqi eye hospitals which might be the main reason behind the poor prognosis of many cases. The purpose of this study was to prepare and pharmaceutically evaluate clotrimazole–β-cyclodextrin (CTZ–β-CD) eyedrops then clinically assess its therapeutic efficacy on fungal keratitis compared with extemporaneous amphotericin B eyedrops (0.5% w/v). A CTZ–β-CD ophthalmic solution was prepared and evaluated by various physicochemical, microbiological, and biological tests. The prepared formula was stable in 0.05 M phosphate buffer pH 7.0 at 40 ± 2°C and 75 ± 5% RH for a period of 6 months. Light has no significant effect on the formula’s stability. The CTZ–β-CD eyedrops efficiently complied with the isotonicity, sterility, and antimicrobiological preservative effectiveness tests. Results of the clinical study revealed that 20 (80%) patients showed a favorable response to the CTZ–β-CD eyedrops, while 16 patients (64%) exhibited a favorable response to amphotericin B (P > 0.05). The mean course of treatment was significantly (P < 0.05) less in the CTZ treatment group than in the amphotericin group (21.5 ± 5.2 vs. 28.3 ± 6.4 days, respectively). The CTZ formulation was significantly (P < 0.05) more effective in the management of severe cases and also against Candida sp. than amphotericin B. There was no significant difference (P < 0.05) between both therapies against filamentous fungi. The CTZ–β-CD formulation can be used alternatively to other ophthalmic antimycotic treatment options in developing countries where stability, cost, or efficacy is a limiting factor.Key words: clotrimazole, β-cyclodextrin, eyedrops, fungal keratitis, Iraq     

Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-016-9945-6) contains supplementary material, which is available to authorized users.     

Quantifying the impact of soil compaction on root system architecture in tomato (Solanum lycopersicum) by X-ray micro-computed tomography

Background and Aims

We sought to explore the interactions between roots and soil without disturbance and in four dimensions (i.e. 3-D plus time) using X-ray micro-computed tomography.

Methods

The roots of tomato Solanum lycopersicum ‘Ailsa Craig’ plants were visualized in undisturbed soil columns for 10 consecutive days to measure the effect of soil compaction on selected root traits including elongation rate. Treatments included bulk density (1·2 vs. 1·6 g cm⁻³) and soil type (loamy sand vs. clay loam).

Key Results

Plants grown at the higher soil bulk density exploited smaller soil volumes (P < 0·05) and exhibited reductions in root surface area (P < 0·001), total root volume (P < 0·001) and total root length (P < 0·05), but had a greater mean root diameter (P < 0·05) than at low soil bulk density. Swelling of the root tip area was observed in compacted soil (P < 0·05) and the tortuosity of the root path was also greater (P < 0·01). Root elongation rates varied greatly during the 10-d observation period (P < 0·001), increasing to a maximum at day 2 before decreasing to a minimum at day 4. The emergence of lateral roots occurred later in plants grown in compacted soil (P < 0·01). Novel rooting characteristics (convex hull volume, centroid and maximum width), measured by image analysis, were successfully employed to discriminate treatment effects. The root systems of plants grown in compacted soil had smaller convex hull volumes (P < 0·05), a higher centre of mass (P < 0·05) and a smaller maximum width than roots grown in uncompacted soil.

Conclusions

Soil compaction adversely affects root system architecture, influencing resource capture by limiting the volume of soil explored. Lateral roots formed later in plants grown in compacted soil and total root length and surface area were reduced. Root diameter was increased and swelling of the root tip occurred in compacted soil.     

Toll-like receptor 4 and high-mobility group box 1 are critical mediators of tissue injury and survival in a mouse model for heatstroke

The molecular mechanisms that initiate the inflammatory response in heatstroke and their relation with tissue injury and lethality are not fully elucidated. We examined whether endogenous ligands released by damaged/stressed cells such as high-mobility group box 1 (HMGB1) signaling through Toll-like receptor 4 (TLR4) may play a pathogenic role in heatstroke. Mutant TLR4-defective (C3H/HeJ) and wild type (C3H/HeOuJ) mice were subjected to heat stress in an environmental chamber pre-warmed at 43.5°C until their core temperature reached 42.7°C, which was taken as the onset of heatstroke. The animals were then allowed to recover passively at ambient temperature. A sham-heated group served as a control. Mutant mice displayed more histological liver damage and higher mortality compared with wild type mice (73% vs. 27%, respectively, P<0.001). Compared to wild type mice, mutant mice exhibited earlier plasma release of markers of systemic inflammation such as HMGB1 (206±105 vs. 63±21 ng/ml; P = 0.0018 and 209±100 vs. 46±32 ng/ml; P<0.0001), IL-6 (144±40 vs. 46±20 pg/ml; P<0.001 and 184±21 vs. 84±54 pg/ml; P = 0.04), and IL-1β (27±4 vs. 1.7±2.3 pg/ml; P<0.0001 at 1 hour). Both strains of mice displayed early release of HMGB1 into the circulation upstream of IL-1β and IL-6 responses which remained elevated up to 24 h. Specific inhibition of HMGB1 activity with DNA-binding A Box (600 µg/mouse) protected the mutant mice against the lethal effect of heat stress (60% A Box vs. 18% GST protein, P = 0.04). These findings suggest a protective role for the TLR4 in the host response to severe heat stress. They also suggest that HMGB1 is an early mediator of inflammation, tissue injury and lethality in heatstroke in the presence of defective TLR4 signaling.     

The in utero programming effect of increased maternal androgens and a direct fetal intervention on liver and metabolic function in adult sheep

Epigenetic changes in response to external stimuli are fast emerging as common underlying causes for the pre-disposition to adult disease. Prenatal androgenization is one such model that results in reproductive and metabolic features that are present in conditions such as polycystic ovary syndrome (PCOS). We examined the effect of prenatal androgens on liver function and metabolism of adult sheep. As non-alcoholic fatty liver disease is increased in PCOS we hypothesized that this, and other important liver pathways including metabolic function, insulin–like growth factor (IGF) and steroid receptivity, would be affected. Pregnant ewes received vehicle control (C; n = 5) or testosterone propionate (TP; n = 9) twice weekly (100 mg; i.m) from d62–102 (gestation 147 days). In a novel treatment paradigm, a second cohort received a direct C (n = 4) or TP (20 mg; n = 7) fetal injection at d62 and d82. In adults, maternal TP exposure resulted in increased insulin secretion to glucose load (P<0.05) and the histological presence of fatty liver (P<0.05) independent of central obesity. Additionally, hepatic androgen receptor (AR; P<0.05), glucocorticoid receptor (GR; P<0.05), UDP- glucose ceramide glucosyltransferase (UGCG; P<0.05) and IGF1 (P<0.01) expression were upregulated. The direct fetal intervention (C and TP) led to early fatty liver changes in all animals without differential changes in insulin secretion. Furthermore, hepatic phosphoenolpyruvate carboxykinase (PEPCK) was up-regulated in the fetal controls (P<0.05) and this was opposed by fetal TP (P<0.05). Hepatic estrogen receptor (ERα; P<0.05) and mitogen activated protein kinase kinase 4 (MAP2K4; P<0.05) were increased following fetal TP exposure. Adult liver metabolism and signaling can be altered by early exposure to sex steroids implicating epigenetic regulation of metabolic disturbances that are common in PCOS.     

A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity

We explored the role of the adiponutrin (PNPLA3) nonsynonymous-rs738409 single nucleotide polymorphism (SNP) in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and whether this SNP contributes to the severity of histological disease. Two hundred sixty-six individuals were evaluated in a case-control association study, which included 172 patients with features of NAFLD and 94 control subjects. The rs738409 G allele was significantly associated with NAFLD (P < 0.001; OR 2.8 95%, CI 1.5–5.2), independent of age, sex, body mass index (BMI), and Homeostasis Model Assessment (HOMA) index. When we tested the hypothesis of a relation between the SNP and the histological spectrum of NAFLD, a significant association was observed [chi2 19.9, degree of freedom (df): 2, P < 5 × 10⁻⁵, adjusted for HOMA and BMI]. The degree of liver steatosis, as evaluated by liver biopsy, was significantly associated with the rs738409 G allele. Patients with CC genotype showed a lower steatosis score (14.9% ± 3.9) in comparison with the CG genotype (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) (P < 0.005). The proportion of the total variation attributed to rs738409 genotypes was 5.3% (β 0.23 ± 0.07; P < 0.002). Our data suggest that the rs738409 G allele is associated not only with fat accumulation in the liver but also with liver injury, possibly triggered by lipotoxicity.     

Exercise reduces cellular stress related to skeletal muscle insulin resistance

This study sought to evaluate the effects of a single session of exercise on the expression of Hsp70, of c-jun N-terminal kinase (JNK), and insulin receptor substrate 1 serine 612 (IRS^ser612) phosphorylation in the skeletal muscle of obese and obese insulin-resistant patients. Twenty-seven volunteers were divided into three experimental groups (eutrophic insulin-sensitive, obese insulin-sensitive, and obese insulin-resistant) according to their body mass index and the presence of insulin resistance. The volunteers performed 60 min of aerobic exercise on a cycle ergometer at 60 % of peak oxygen consumption. M. vastus lateralis samples were obtained before and after exercise. The protein expressions were evaluated by Western blot. Our findings show that compared with paired eutrophic controls, obese subjects have higher basal levels of p-JNK (100 ± 23 % vs. 227 ± 67 %, p = 0.03) and p-IRS-1^ser612 (100 ± 23 % vs. 340 ± 67 %, p < 0.001) and reduced HSP70 (100 ± 16 % vs. 63 ± 12 %, p < 0.001). The presence of insulin resistance results in a further increase in p-JNK (460 ± 107 %, p < 0.001) and a decrease in Hsp70 (46 ± 5 %, p = 0.006), but p-IRS-1^ser612 levels did not differ from obese subjects (312 ± 73 %, p > 0.05). Exercise reduced p-JNK in obese insulin-resistant subjects (328 ± 33 %, p = 0.001), but not in controls or obese subjects. Furthermore, exercise reduced p-IRS-1^ser612 for both obese (122 ± 44 %) and obese insulin-resistant (185 ± 36 %) subjects. A main effect of exercise was observed in HSP70 (p = 0.007). We demonstrated that a single session of exercise promotes changes that characterize a reduction in cellular stress that may contribute to exercise-induced increase in insulin sensitivity.     

BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10⁴ gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.