Current Advancements in Mouse Models of Retinal Disease.

The field of retinal degenerative (RDs) disease study has been in a state of exponential growth from discovering the underlying genetic components of such diseases as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) to the first gene therapy developed and approved for human Leber congenital amaurosis. However, a source for high-fidelity animal models of these complex, multifactorial, and/or polygenic diseases is a need that has yet to be fulfilled. While models for AMD and RP do exist, they often require aging the animals for a year or more, feeding special diets, or introduction of external modulators such as exposure to cigarette smoke. Currently, work is being done to uncover high-fidelity naturally occurring models of these retinal diseases with the hope and intent of providing the vision community the tools it needs to better understand, treat, and, one day, cure the patients suffering from these devastating afflictions.

P-glycoprotein Restricts Ocular Penetration of Loperamide across the Blood-Ocular Barriers: a Comparative Study in Mdr1a Knock-out and Wild Type Sprague Dawley Rats.

The current research was undertaken to determine the existence and magnitude of P-glycoprotein (P-gp) expression on the blood-ocular barriers by studying the ocular penetration of loperamide, a specific P-gp substrate, in P-gp (Mdr1a) knock-out (KO) and wild type (WT) Sprague Dawley rats. A clear, stable, sterile solution of loperamide (1 mg/mL), for intravenous administration, was formulated and evaluated. Ocular distribution was studied in P-gp KO and WT rats following intravenous administration of loperamide (at two doses). The drug levels in plasma, aqueous humor (AH), and vitreous humor (VH) samples were determined with the aid of UHPLC-Q-TOF-MS/MS, and the AH/plasma (D AH ) and VH/plasma (D VH ) distribution ratios were estimated. Electroretinography (ERG), ultrastructural analyses, and histology studies were carried out, in both KO and WT rats, to detect any drug-induced functional and/or structural alterations in the retina. Dose-related loperamide levels were observed in the plasma of both WT and KO rats. The loperamide concentrations in the AH and VH of KO rats were significantly higher compared to that observed in the WT rats, at the lower dose. However, a marked increase in the D AH and D VH was noted in the KO rats. ERG, ultrastructure, and histology studies did not indicate any drug-induced toxic effects in the retina under the test conditions. The results from these studies demonstrate that P-gp blocks the penetration of loperamide into the ocular tissues from the systemic circulation and that the effect is more pronounced at lower plasma loperamide concentrations.

A crystallin gene network in the mouse retina.

The present study was designed to examine the regulation of crystallin genes and protein in the mouse retina using the BXD recombinant inbred (RI) strains. Illumina Sentrix BeadChip Arrays (MouseWG-6v2) were used to analyze mRNA levels in 75 BXD RI strains along with the parental strains (C57Bl/6J and DBA/2J), and the reciprocal crosses in the Hamilton Eye Institute (HEI) Retina Dataset (www.genenetwork.org). Protein levels were investigated using immunoblots to quantify levels of proteins and indirect immunohistochemistry to define the distribution of protein. Algorithms in the Genomatix program were used to identify transcription factor binding sites common to the regulatory sequences in the 5' regions of co-regulated set of crystallin and other genes as compared to a set of control genes. As subset of genes, including many encoding lens crystallins is part of a tightly co-regulated network that is active in the retina. Expression of this crystallin network appears to be binary in nature, being expressed either at relatively low levels or being highly upregulated. Relative to a control set of genes, the 5' regulatory sequences of the crystallin network genes show an increased frequency of a set of common transcription factor-binding sites, the most common being those of the Maf family. Chromatin immunoprecipitation of human lens epithelial cells (HLEC) and rat retinal ganglion cells (RGC) confirmed the functionality of these sites, showing that MafA binds the predicted sites of CRYGA and CRYGD in HLE and CRYAB, CRYGA, CRYBA1, and CRYBB3 in RGC cells. In the retina there is a highly correlated group of genes containing many members of the α- ß- and γ-crystallin families. These genes can be dramatically upregulated in the retina. One transcription factor that appears to be involved in this coordinated expression is the MAF family transcription of factors associated with both lens and extralenticular expression of crystallin genes.

EDL-291, a novel isoquinoline, presents antiglioblastoma effects in vitro and in vivo.

To investigate the effectiveness of EDL-291, a 6,7-dimethoxy-1-[4-(4-methoxypyridin-3-yl)benzyl]-1,2,3,4-tetrahydroisoquinoline dihydrochloride compound, in inhibiting the survival of glioblastoma in vitro and in vivo. Dose-response curves were generated to determine the EC50 in rat and human glioblastoma cell lines by treatment with different dilutions of EDL-291. To evaluate the architecture of the glioblastoma cells after treatment with EDL-291, the rat and human glioblastoma cells were stained with Mito Tracker Green FM. To determine whether autophagy was induced in EDL-291-treated glioblastoma cells, both rat and human glioblastoma cell lines were stained with acridine orange and light chain-3 immunoblots were performed. The efficacy of EDL-291 was monitored in vivo using a rat glioblastoma model. Rat glioblastoma cells were transplanted into an intracranial rat model, followed by infusions of saline, a low dose of EDL-291 (20 mg/kg for the first half hour, followed by 40 mg/kg EDL-291 in saline for 4 h), or a high dose of EDL-291 (60 mg/kg for the first half hour, followed by 90 mg/kg EDL-291 for 4 h). EDL-291 inhibits glioblastoma in vitro by destroying the mitochondria as shown with Mito Tracker Green FM. Acridine orange staining and light chain-3 immunoblots suggest that autophagy is induced when glioblastoma cells are treated with EDL-291. In vivo, a low dosage of EDL-291 is sufficient and effective in reducing glioblastoma tumor size. EDL-291 selectively induces cell death in rat and human glioblastoma cell lines by the induction of autophagy. EDL-291 exhibits antiglioblastoma effects both in vitro and in vivo.

Chronic Proinflammatory Signaling Accelerates the Rate of Degeneration in a Spontaneous Polygenic Model of Inherited Retinal Dystrophy.

Collectively, retinal neurodegenerative diseases are comprised of numerous subtypes of disorders which result in loss of a varying cell types in the retina. These diseases can range from glaucoma, which results in retinal ganglion cell death, to age-related macular degeneration and retinitis pigmentosa, which result in cell death of the retinal pigment epithelium, photoreceptors, or both. Regardless of the disease, it's been recently found that increased release of proinflammatory cytokines and proliferation of active microglia result in a remarkably proinflammatory microenvironment that assists in the pathogenesis of the disease; however, many of the details of these inflammatory events have yet to be elucidated. In an ongoing study, we have used systems genetics to identify possible models of spontaneous polygenic age-related macular degeneration by mining the BXD family of mice using single nucleotide polymorphism analyses of known genes associated with the human retinal disease. One BXD strain (BXD32) was removed from the study as the rate of degeneration observed in these animals was markedly increased with a resultant loss of most all photoreceptors by 6 months of age. Using functional and anatomical exams including optokinetic nystamography, funduscopy, fluorescein angiography, and optical coherence tomography, along with immunohistochemical analyses, we show that the BXD32 mouse strain exhibits a severe neurodegenerative phenotype accompanied by adverse effects on the retinal vasculature. We also expose the concurrent establishment of a chronic proinflammatory microenvironment including the TNFα secretion and activation of the NF-κB and JAK/STAT pathways with an associated increase in activated macrophages and phagoptosis. We conclude that the induced neuronal death and proinflammatory pathways work synergistically in the disease pathogenesis to enhance the rate of degeneration in this spontaneous polygenic model of inherited retinal dystrophy.

Proinflammatory Pathways Are Activated in the Human Q344X Rhodopsin Knock-In Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.

Enhanced Corneal Penetration of a Poorly Permeable Drug Using Bioadhesive Multiple Microemulsion Technology.

Corneal penetration is a key rate limiting step in the bioavailability of topical ophthalmic formulations that incorporate poorly permeable drugs. Recent advances have greatly aided the ocular delivery of such drugs using colloidal drug delivery systems. Ribavirin, a poorly permeable BCS class-III drug, was incorporated in bioadhesive multiple W/O/W microemulsion (ME) to improve its corneal permeability. The drug-loaded ME was evaluated regarding its physical stability, droplet size, PDI, zeta potential, ultrastructure, viscosity, bioadhesion, in vitro release, transcorneal permeability, cytotoxicity, safety and ocular tolerance. Our ME possessed excellent physical stability, as it successfully passed several cycles of centrifugation and freeze-thaw tests. The formulation has a transparent appearance due to its tiny droplet size (10 nm). TEM confirmed ME droplet size and revealed its multilayered structure. In spite of the high aqueous solubility and the low permeability of ribavirin, this unique formulation was capable of sustaining its release for up to 24 h and improving its corneal permeability by 3-fold. The in vitro safety of our ME was proved by its high percentage cell viability, while its in vivo safety was confirmed by the absence of any sign of toxicity or irritation after either a single dose or 14 days of daily dosing. Our ME could serve as a vehicle for enhanced ocular delivery of drugs with different physicochemical properties, including those with low permeability.

Once Daily Pregabalin Eye Drops for Management of Glaucoma.

Elevated intraocular pressure (IOP) is the most significant risk factor contributing to visual field loss in glaucoma. Unfortunately, the deficiencies associated with current therapies have resulted in reduced efficacy, several daily dosings, and poor patient compliance. Previously, we identified the calcium voltage-gated channel auxiliary subunit alpha2delta 1 gene (Cacna2d1) as a modulator of IOP and demonstrated that pregabalin, a drug with high affinity and selectivity for CACNA2D1, lowered IOP in a dose-dependent manner. Unfortunately, IOP returned to baseline at 6 h after dosing. In the current study, we develop a once daily topical pregabalin-loaded multiple water-in-oil-in-water microemulsion formulation to improve drug efficacy. We characterize our formulations using multiple in vitro and in vivo evaluations. Our lead formulation provides continuous release of pregabalin for up to 24 h. Because of its miniscule droplet size (<20 nm), our microemulsion has a transparent appearance and should not blur vision. It is also stable at one month of storage at temperatures ranging from 5 to 40 °C. Our formulation is nontoxic, as illustrated by a cell toxicity study and slit-lamp biomicroscopic exams. CACNA2D1 is highly expressed in both the ciliary body and the trabecular meshwork, where it functions to modulate IOP. A single drop of our lead pregabalin formulation reduces IOP by greater than 40%, which does not return to baseline until >30 h post-application. Although there were no significant differences in the amplitude of IOP reduction between the formulations we tested, a significant difference was clearly observed in their duration of action. Our multilayered microemulsion is a promising carrier that sustains the release and prolongs the duration of action of pregabalin, a proposed glaucoma therapeutic.

NA3 glycan: a potential therapy for retinal pigment epithelial deficiency.

Atrophic age-related macular degeneration (AMD) is the most common type of AMD, yet there is no United States Food and Drug Administration (FDA)-approved therapy. This disease is characterized by retinal pigment epithelial (RPE) insufficiency, primarily in the macula, which affects the structure and physiology of photoreceptors and ultimately, visual function. In this study, we evaluated the protective effects of a naturally derived small molecule glycan therapeutic-asialo-, tri-antennary complex-type N-glycan (NA3)-in two distinct preclinical models of atrophic AMD. In RPE-deprived Xenopus laevis tadpole eyes, NA3 supported normal retinal ultrastructure. In RCS rats, NA3 supported fully functioning visual integrity. Furthermore, structural analyses revealed that NA3 prevented photoreceptor outer segment degeneration, pyknosis of the outer nuclear layer, and reactive gliosis of Müller cells (MCs). It also promoted maturation of adherens junctions between MC and photoreceptors. Our results demonstrate the neuroprotective effects of a naturally derived small molecular glycan therapeutic-NA3-in two unique preclinical models with RPE insufficiency. These data suggest that NA3 glycan therapy may provide a new therapeutic avenue in the prevention and/or treatment of retinal diseases such as atrophic AMD.

Plasma and cerebrospinal fluid pharmacokinetics of the novel tetrahydroisoquinoline EDL-155 in rats.

PURPOSE: Tetrahydroisoquinolines (THIs) have demonstrated anti-cancer activity in rodent models of glioma, a form of brain cancer refractory to therapeutic intervention. In this study, peripheral and cerebrospinal fluid (CSF) pharmacokinetics in rats were determined to assess the drug developability of the novel THI EDL-155 for the treatment of glioma. METHODS: Serial blood and CSF samples were collected from rats following intravenous bolus administration of EDL-155 (10-20 mg/kg). Samples were analyzed by LC/MS/MS. Pharmacokinetic analyses using compartmental and noncompartmental methods were performed using the computer program WinNonlin. Plasma protein binding was measured using the charcoal adsorption method. The in vivo efficacy of EDL-155 (i.p. 20 mg/kg twice daily for 7 days) was assessed in rats with stereotactically implanted C6 glioma cells into the caudate. RESULTS: EDL-155 plasma concentration data were described by a one-compartment model. EDL-155 demonstrated rapid clearance (342.5+/-49.9 ml/min/kg), high volume of distribution (13.0+/-1.2 l/kg) and a terminal half-life of 23.7+/-1.5 min. Dose-normalized CSF area under the curve (AUC(CSF)) as a percentage of peripheral exposure (AUC(Plasma)) was 1.4%. EDL-155 was highly bound to plasma proteins (>93%). Intracranial tumor volume at 7 days post-implantation was approximately 30% smaller in animals treated with EDL-155 when compared to vehicle control animals (13.2+/-5.3 mm(3) vs. 18.7+/-6.3 mm(3); P=0.04). CONCLUSION: High clearance and extensive protein binding limit the brain availability of EDL-155 following systemic administration. EDL-155 treatment resulted in reduced tumor size despite limited blood brain barrier penetrability, which suggests that analogs with increased metabolic stability and brain penetrability may provide a therapeutic option for primary central nervous system tumors such as glioma. On-going studies are focused on the design, synthesis, and testing of novel analogs based upon these findings.

Retina Compatible Interactions and Effective Modulation of Blood Ocular Barrier P-gp Activity by Third-Generation Inhibitors Improve the Ocular Penetration of Loperamide.

Effective drug delivery to the deeper ocular tissues remains an unresolved conundrum mainly due to the expression of multidrug resistance efflux proteins, besides tight junction proteins, in the blood ocular barriers (BOBs). Hence, the purpose of the current research was to investigate the ability of the third-generation efflux protein inhibitors, elacridar (EQ), and tariquidar (TQ), to diminish P-glycoprotein (P-gp) mediated efflux transport of loperamide (LOP), a P-gp substrate, across the BOB in Sprague Dawley rats. Initially, Western blot analysis confirmed the expression of P-gp in the iris-ciliary bodies and the retina choroid in the wild type rats. Next, the ocular distribution of LOP, in the presence and absence of EQ/TQ (at 2 doses), was evaluated. The significantly higher aqueous humor/plasma (DAH) and vitreous humor (VH)/plasma (DVH) distribution ratios of LOP in the rats pretreated with EQ or TQ demonstrated effective inhibition of P-gp activity in the BOB. Interestingly, the modulation of P-gp activity by EQ/TQ was more pronounced at the lower dose. The normal functioning and architecture of the retina, as indicated by electroretinography studies, confirmed the cytocompatibility of LOP and EQ/TQ interactions at the doses tested.

The developmental regulation of CD81 in the rat retina.

PURPOSE: The tetraspanin CD81 is expressed in Müller glial cells and retinal pigment epithelium (RPE). CD81 and other members of the tetraspanin family link extracellular interactions of cells into intracellular cascades. This study examined the developmental expression of CD81 and protein-protein interactions linking CD81 to intracellular proteins. METHODS: We used synthetic peptides of the C-terminal intracellular domains of CD81 to probe fusion proteins of PDZ domains blotted to nitrocellulose membranes, then confirmed the relationships between the PDZ proteins using immunoprecipitation methods. Colocalization of the associated proteins was analyzed across development, using double-label immunohistochemical methods in the retina of Sprague-Dawley rats. RESULTS: The C-terminal intracellular sequences of CD81 bound to three putative PDZ domains that potentially represented domains on Sap97 and EBP50. In immunoprecipitation experiments using RPE cells, CD81 coprecipitated with both proteins, EBP50 and Sap97. Like CD81, EBP50 and Sap97 are expressed at low levels immediately after birth and upregulated during the first two postnatal weeks, reaching almost adult levels at postnatal day 20. In the RPE layer, synapse-associated protein 97 (Sap97) and CD81 were associated with the basolateral surface of the cells; ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) localizing with CD81 was found on microvilli at the inner surface of RPE cells. CONCLUSIONS: These results support the hypothesis that CD81 is associated with the final stages of RPE cell maturation, establishing key molecular interactions linking the cell membrane proteins into macromolecular complexes containing PDZ protein scaffolds.

Vitamin B6 protects primate retinal neurons from ischemic injury.

Vitamin B6 derivatives protect the retinal neurons from excitotoxic injury in vitro. However, their in vivo role in a process involving excitotoxicity, such as ischemia, remains unknown. We studied potential protective effects of pyridoxal 5'-phosphate (PLP) and pyridoxal hydrochloride (pyridoxal) on the retinal neurons in a monkey model of transient global ischemia. Daily intravenous injections (15 mg/kg) of pyridoxal and PLP were performed for consecutive 10 days. On the sixth day, whole brain complete ischemia was produced by clipping the innominate and the left subclavian arteries for 20 min. The monkeys were sacrificed 5 days after ischemia and their retinas were processed for histological analysis. The ischemia induced a marked cellular injury in the retina as shown by the loss of ganglion cells and the reduction of thickness of the ganglion cell, inner plexiform, and inner nuclear layers. PLP significantly prevented the ganglion cell loss and the reduction of thickness of the ganglion cell layer. Pyridoxal significantly prevented the ganglion cell loss as well as the reduction of thickness of ganglion cell, inner plexiform and inner nuclear layers. These results suggest that PLP and pyridoxal counteract the postischemic neuronal death in the adult primate retina, offering a potential for a novel pharmacotherapy of retinal ischemic injury.

Synthesis and in vitro evaluation of novel 1,2,3,4-tetrahydroisoquinoline derivatives as potent antiglioma agents.

Glioblastoma Multiforme (GBM) continues to demand improved chemotherapeutic solutions. In order to discover novel chemotherapeutic agents for GBM, we identified novel tetrahydroisoquinoline (THI) analogs as antiglioma agents. The present study reports the design, synthesis and in vitro evaluation of new THI derivatives in four established human glioma cell lines (T98, U87, LN18 and A172). Our structure activity relationship (SAR) studies revealed that the important modification of the carbon linker between the biphenyl and THI ring yielded EDL-360 (12) as a potent antiglioma agent (LN18; IC50: 5.42 ± 0.06 µM) and is considered to be our new lead drug candidate for further preclinical studies.

Treating retinoblastoma in tissue culture and in a rat model with a novel isoquinoline derivative.

PURPOSE. To investigate the effectiveness of a novel isoquinoline derivative, EDL-155, in killing retinoblastoma in vitro and in vivo. METHODS. Dose-response curves were generated in which Y79 retinoblastoma cells tagged with luciferase (Y79-Luc) were treated with serial concentrations of EDL-155. Electron microscopy was used to evaluate the ultrastructural morphology of EDL-155-treated Y79 cells. To determine whether autophagy was induced in EDL-155-treated Y79-Luc cells, staining with acridine orange and LC-3 immunoblot analysis was performed. To evaluate the efficacy of EDL-155 in vivo, Y79-Luc retinoblastoma cells were injected into the vitreous cavity of newborn rats, followed by periocular injections of EDL-155 (20 mg/kg/day) or an equivalent dosage of saline. RESULTS. EDL-155 appeared to destroy the retinoblastoma cells in vitro with an EC(50) of 9.1 micriM. EDL-155-treated retinoblastoma cells displayed a lack of viable mitochondria and the presence of autophagosomes wrapped in the characteristic double membranes. Acridine orange staining of EDL-155-treated retinoblastoma cells demonstrated the accumulation of vacuoles, and the immunoblots displayed a shift in molecular weight of LC-3, indicative of incorporation into autophagosome vesicles. In the retinoblastoma animal model, four doses of EDL-155 were delivered over 4 days, which was sufficient to see a significant decrease (P = 0.01) in viable intraocular tumors. Seven of the 25 rats treated with EDL-155 had no detectable living tumor. No significant decrease in viable tumor was observed in control animals. CONCLUSIONS. EDL-155 appears to eliminate retinoblastoma cells by disrupting mitochondria and inducing autophagy. Local delivery of EDL-155 may be an effective therapy for some types of ocular cancers.

Fast and sensitive liquid chromatography/electrospray mass spectrometry method to study ocular penetration of EDL-155, a novel antitumor agent for retinoblastoma in rats.

Our group has used the tetrahydroisoquinoline derivative EDL-155 to treat glioblastoma in animal models and it is currently being evaluated in the treatment of ocular cancers. The purpose of this study was to develop a rapid and sensitive liquid chromatography and tandem mass spectrometry (LC-MS/MS) method to study the plasma and vitreous humor disposition of EDL-155 in rats. Animals received a single periocular injection of EDL-155 (20 mg/kg). Animals were sacrificed at specified times (5, 60, 120, 240 and 360 min) and plasma and vitreous humor samples were obtained. EDL-155 was isolated by protein precipitation and the extracts were analyzed by reversed-phase high-pressure liquid chromatography (HPLC) with MS/MS detection. A structurally similar analog was used as internal standard (IS). The chromatographic run time was 3.5 min per injection. The mass spectrometer was operated in positive-ion, multiple reaction monitoring (MRM) mode. The mass transitions monitored were m/z332.2 --> 167.2 (EDL-155) and m/z391.2 --> 200.2 (IS). The lower limit of quantification (LLOQ) was 0.1 ng/ml in both vitreous humor and plasma. The method was validated for selectivity, linearity, accuracy and precision in rat vitreous humor and partially validated for accuracy and precision in rat plasma. The ion suppression, recovery and stability of the analyte in the biological matrix were also tested. The assay was rapid, sensitive and robust enough to support EDL-155 ocular penetration studies in a rodent model of intraocular cancer. Application of this method revealed that EDL-155 was rapidly passed into the vitreous humor following periocular administration. Further, vitreous humor exposure exceeded systemic exposure by approximately sevenfold. High local concentrations coupled with minimal systemic exposure supports further testing of EDL-155 as localized therapy for intraocular cancers.

Cardiac stem cells in brown adipose tissue express CD133 and induce bone marrow nonhematopoietic cells to differentiate into cardiomyocytes.

Recently, there has been noteworthy progress in the field of cardiac regeneration therapy. We previously reported that brown adipose tissue (BAT) contained cardiac progenitor cells that were relevant to the regeneration of damaged myocardium. In this study, we found that CD133-positive, but not c-Kit- or Sca-1-positive, cells in BAT differentiated into cardiomyocytes (CMs) with a high frequency. Moreover, we found that CD133(+) brown adipose tissue-derived cells (BATDCs) effectively induced bone marrow cells (BMCs) into CMs. BMCs are considered to have the greatest potential as a source of CMs, and two sorts of stem cell populations, the MSCs and hematopoietic stem cells (HSCs), have been reported to differentiate into CMs; however, it has not been determined which population is a better source of CMs. Here we show that CD133-positive BATDCs induce BMCs into CMs, not through cell fusion but through bivalent cation-mediated cell-to-cell contact when cocultured. Moreover, BMCs induced by BATDCs are able to act as CM repletion in an in vivo infarction model. Finally, we found that CD45(-)CD31(-) CD105(+) nonhematopoietic cells, when cocultured with BATDCs, generated more than 20 times the number of CMs compared with lin(-)c-Kit(+) HSCs. Taken together, these data suggest that CD133-positive BATDCs are a useful tool as CM inducers, as well as a source of CMs, and that the nonhematopoietic fraction in bone marrow is also a major source of CMs. Disclosure of potential conflicts of interest is found at the end of this article.

[Molecule character of rabies virus in 3 counties in Hunan China].

OBJECTIVE: To study the rabies molecular biology features in animals between high incidence area of rabies and no rabies cases area in Hunan. METHODS: detect saliva of dogs and brains of dogs and cats by direct immunofluorescence assay, review positive samples by RT-PCR, sequencing extract RNA virus for genetic analysis. RESULTS: 12 were detected rabies virus antigen and positive nucleoside acid in 82 dogs from Wugang city also 1 in 17 from Dongkou county; the positive rate: Wugang 14.63 percent, Dongkou 5.88 percent. No rabies virus was detected in 67 samples of dog brains from Fenghuang County. Also none in 28 samples of cat brains. Amplificating N gene of rabies virus from positive samples of dog brain's tissue (No Wg13, Dk13) by RT-PCR, it shows that homology of nucleoside acid between two strain of virus is 99.4 percent; also 99.1 percent of amino acid. The homology of nucleoside acid (amonio acid) among Wg13 stain and Chinese strain CTN and aG strain is 89.4 percent (98.2 percent) and 86.1 percent (95.1 percent); The homology of nucleoside acid (amonio acid) among Dk13 stain Chinese strain CTN and aG strain is 89.1 percent (98.0 percent), 86.1 percent (94.9 percent).Compare with isolated rabies virus from abroad, the homology between two strains and Indonesia is 92.8 percent and 93.2 percent, the most similar of them. The strains isolated from other countries including Japan, Sri Lanka and India are relatively lower; The sequence of gene Wg13 and Dk13 were taken replacement of amino acid. CONCLUSION: Two strains are belong to type I rabies virus, comparing its N gene with current using vaccine strains, both are in same group, and homology are relatively higher.

Cardiac progenitor cells in brown adipose tissue repaired damaged myocardium.

Cardiomyocyte (CM) regeneration is limited in adult life and is not sufficient to prevent myocardial infarction. Hence, the identification of a useful source of CM progenitors is of great interest for possible use in regenerative therapy. Mesenchymal stem cells in bone marrow, embryonic stem cells, and skeletal myoblasts are known sources of CM repletion; however, there are a number of critical problems for clinical application. In this study, we succeeded to identify CM progenitor cells in brown adipose tissue (BAT). Moreover, we showed that CM progenitor cells in BAT that existed in CD29-positive population could differentiate into CM with high efficiency. To confirm the in vivo effect of CD29(+)BAT-derived cells (BATDCs), we transplanted these cells into infarct border zone of an acute myocardial infarction model in rat. Results clearly indicated that implantation of CD29(+) BATDCs led to the reduction of the infarction area and improvement of left ventricular function by replacing newly developed CMs in comparison with that by CD29(+) white adipose tissue-derived cells or control saline. These findings suggest that BATDCs are one of the useful sources for a new strategy in CM regeneration.

[Analysis on the epidemiology of rabies in Hunan province in the past 10 years].

OBJECTIVE: To explore the possible factors causing the increase of rabies cases and to provide references for the development of related prevention and control strategy in Hunan. METHODS: Data was collected and analyzed on epidemic situation of rabies in the past ten years, and studies were carried in some counties. RESULTS: From 1994 to 1999, the annual cases sustained between 17 and 78. However, the number of cases has continued to increase since 2000. The annual reported cases in 2001 and 2002 were 311 and 313, and accounted for 34.8% and 30.0% of the total cases in the whole country respectively. The epidemic areas were mainly located in the south and middle parts of Hunan. Furthermore, the epidemic areas had been expanding. In 1997, human rabies cases were only reported in 7 cities but increased to 12 cities in 2004. CONCLUSION: Factors as increasing number but low inoculating rate to dogs, and incorrect treatment on the wounds etc. might have served important roles for the recurrence of rabies. Hence, it is necessary to take comprehensive preventive measures to control and prevent the epidemics of rabies in Hunan.