The Beneficial Effect of Suramin on Monocrotaline-Induced Pulmonary Hypertension in Rats

Background

Pulmonary hypertension (PH) is a progressive disorder characterized by an increase in pulmonary artery pressure and structural changes in the pulmonary vasculature. Several observations indicate that growth factors play a key role in PH by modulating pulmonary artery smooth muscle cell (PA-SMC) function. In rats, established monocrotaline-induced PH (MCT-PH) can be reversed by blocking platelet-derived growth factor receptors (PDGF-R), epidermal growth factor receptors (EGF-R), or fibroblast growth factor receptors (FGF-R). All these receptors belong to the receptor tyrosine kinase (RTK) family.

Methods and Results

We evaluated whether RTK blockade by the nonspecific growth factor inhibitor, suramin, reversed advanced MCT-PH in rats via its effects on growth-factor signaling pathways. We found that suramin inhibited RTK and ERK1/2 phosphorylation in cultured human PA-SMCs. Suramin inhibited PA-SMC proliferation induced by serum, PDGF, FGF2, or EGF in vitro and ex vivo. Treatment with suramin from day 1 to day 21 after monocrotaline injection attenuated PH development, as shown by lower values for pulmonary artery pressure, right ventricular hypertrophy, and distal vessel muscularization on day 21 compared to control rats. Treatment with suramin from day 21 to day 42 after monocrotaline injection reversed established PH, thereby normalizing the pulmonary artery pressure values and vessel structure. Suramin treatment suppressed PA-SMC proliferation and attenuated both the inflammatory response and the deposition of collagen.

Conclusions

RTK blockade by suramin can prevent MCT-PH and reverse established MCT-PH in rats. This study suggests that an anti-RTK strategy that targets multiple RTKs could be useful in the treatment of pulmonary hypertension.     

Tuberculosis Screening by Tuberculosis Skin Test or QuantiFERON?-TB Gold In-Tube Assay among an Immigrant Population with a High Prevalence of Tuberculosis and BCG Vaccination

Rationale

Each year 1 million persons acquire permanent U.S. residency visas after tuberculosis (TB) screening. Most applicants undergo a 2-stage screening with tuberculin skin test (TST) followed by CXR only if TST-positive at > 5 mm. Due to cross reaction with bacillus Calmette-Guérin (BCG), TST may yield false positive results in BCG-vaccinated persons. Interferon gamma release assays exclude antigens found in BCG. In Vietnam, like most high TB-prevalence countries, there is universal BCG vaccination at birth.

Objectives

1. Compare the sensitivity of QuantiFERON ®-TB Gold In-Tube Assay (QFT) and TST for culture-positive pulmonary TB. 2. Compare the age-specific and overall prevalence of positive TST and QFT among applicants with normal and abnormal CXR.

Methods

We obtained TST and QFT results on 996 applicants with abnormal CXR, of whom 132 had TB, and 479 with normal CXR.

Results

The sensitivity for tuberculosis was 86.4% for QFT; 89.4%, 81.1%, and 52.3% for TST at 5, 10, and 15 mm. The estimated prevalence of positive results at age 15–19 years was 22% and 42% for QFT and TST at 10 mm, respectively. The prevalence increased thereafter by 0.7% year of age for TST and 2.1% for QFT, the latter being more consistent with the increase in TB among applicants.

Conclusions

During 2-stage screening, QFT is as sensitive as TST in detecting TB with fewer requiring CXR and being diagnosed with LTBI. These data support the use of QFT over TST in this population.      

Prefabrication of 3D Cartilage Contructs: Towards a Tissue Engineered Auricle – A Model Tested in Rabbits

The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15×8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.     

Primary Drug-Resistant Tuberculosis in Hanoi,Viet Nam: Present Status and Risk Factors

Introduction

Resistance of Mycobacterium tuberculosis (MTB) to anti-tuberculosis (TB) drugs presents a serious challenge to TB control worldwide. We investigated the status of drug resistance, including multidrug-resistant (MDR) TB, and possible risk factors among newly diagnosed TB patients in Hanoi, the capital of Viet Nam.

Methods

Clinical and epidemiological information was collected from 506 newly diagnosed patients with sputum smear- and culture-positive TB, and 489 (96.6%) MTB isolates were subjected to conventional drug susceptibility testing, spoligotyping, and 15-locus variable numbers of tandem repeats typing. Adjusted odds ratios (aORs) were calculated to analyze the risk factors for primary drug resistance.

Results

Of 489 isolates, 298 (60.9%) were sensitive to all drugs tested. Resistance to isoniazid, rifampicin, streptomycin, ethambutol, and MDR accounted for 28.2%, 4.9%, 28.2%, 2.9%, and 4.5%, respectively. Of 24 isolates with rifampicin resistance, 22 (91.7%) were MDR and also resistant to streptomycin, except one case. Factors associated with isoniazid resistance included living in old urban areas, presence of the Beijing genotype, and clustered strains [aOR = 2.23, 95% confidence interval (CI) 1.15–4.35; 1.91, 1.18–3.10; and 1.69, 1.06–2.69, respectively). The Beijing genotype was also associated with streptomycin resistance (aOR = 2.10, 95% CI 1.29–3.40). Human immunodeficiency virus (HIV) coinfection was associated with rifampicin resistance and MDR (aOR = 5.42, 95% CI 2.07–14.14; 6.23, 2.34–16.58, respectively).

Conclusion

Isoniazid and streptomycin resistance was observed in more than a quarter of TB patients without treatment history in Hanoi. Transmission of isoniazid-resistant TB among younger people should be carefully monitored in urban areas, where Beijing strains and HIV coinfection are prevalent. Choosing an optimal treatment regimen on the basis of the results of drug susceptibility tests and monitoring of treatment adherence would minimize further development of drug resistance strains.     

Detection of Amyloid Plaques Targeted by Bifunctional USPIO in Alzheimer’s Disease Transgenic Mice Using Magnetic Resonance Microimaging

Amyloid plaques are a key pathological hallmark of Alzheimer’s disease (AD). The detection of amyloid plaques in the brain is important for the diagnosis of AD, as well as for following potential amyloid targeting therapeutic interventions. Our group has developed several contrast agents to detect amyloid plaques in vivo using magnetic resonance microimaging (µMRI) in AD transgenic mice, where we used mannitol to enhance blood brain barrier (BBB) permeability. In the present study, we used bifunctional ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Aβ1-42 peptide to image amyloid plaque deposition in the mouse brain. We coupled the nanoparticles to polyethylene glycol (PEG) in order to improve BBB permeability. These USPIO-PEG-Aβ1-42 nanoparticles were injected intravenously in AD model transgenic mice followed by initial in vivo and subsequent ex vivo μMRI. A 3D gradient multi-echo sequence was used for imaging with a 100 µm isotropic resolution. The amyloid plaques detected by T2*-weighted μMRI were confirmed with matched histological sections. The region of interest-based quantitative measurement of T2* values obtained from the in vivo μMRI showed contrast injected AD Tg mice had significantly reduced T2* values compared to wild-type mice. In addition, the ex vivo scans were examined with voxel-based analysis (VBA) using statistical parametric mapping (SPM) for comparison of USPIO-PEG-Aβ1-42 injected AD transgenic and USPIO alone injected AD transgenic mice. The regional differences seen by VBA in the USPIO-PEG-Aβ1-42 injected AD transgenic correlated with the amyloid plaque distribution histologically. Our results indicate that USPIO-PEG-Aβ1-42 can be used for amyloid plaque detection in vivo by intravenous injection without the need to co-inject an agent which increases permeability of the BBB. This technique could aid the development of novel amyloid targeting drugs by allowing therapeutic effects to be followed longitudinally in model AD mice.     

Dextromethorphan attenuates trimethyltin-induced neurotoxicity via sigma1 receptor activation in rats

We showed that dextromethorphan (DM) provides neuroprotective/anticonvulsant effects and that DM and its major metabolite, dextrorphan, have a high-affinity for sigma(1) receptors, but a low affinity for sigma(2) receptors. In addition, we found that DM has a higher affinity than DX for sigma(1) sites, whereas DX has a higher affinity than DM for PCP sites. We extend our earlier findings by showing that DM attenuated trimethyltin (TMT)-induced neurotoxicity (convulsions, hippocampal degeneration and spatial memory impairment) in rats. This attenuation was reversed by the sigma(1) receptor antagonist BD 1047, but not by the sigma(2) receptor antagonist ifenprodil. DM attenuates TMT-induced reduction in the sigma(1) receptor-like immunoreactivity of the rat hippocampus, this attenuation was blocked by the treatment with BD 1047, but not by ifenprodil. These results suggest that DM prevents TMT-induced neurotoxicity, at least in part, via sigma(1) receptor stimulation.     

Limitation of current probe design for oligo-cross-FISH,exemplified by chromosome evolution studies in duckweeds

Chromosoma - Duckweeds represent a small, free-floating aquatic family (Lemnaceae) of the monocot order Alismatales with the fastest growth rate among flowering plants. They comprise five genera...     

Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC’s undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.