Comparison of effectiveness between transthoracic echocardiography and X-ray guided closure of patent foramen ovale: A retrospective analysis.

Objective: To compare the effectiveness of transthoracic echocardiography (TTE) and X-ray guided closure of patent foramen ovale (PFO). Methods: In this retrospective study, clinical data from 90 patients who underwent PFO occlusion surgery in the First People's Hospital of Yongkang from January 2020 to December 2022 were retrospectively reviewed. Among them, 43 patients underwent X-ray guided PFO occlusion surgery (X-ray group) while 47 patients underwent TTE guided PFO occlusion surgery (TTE group). Perioperative, cardiac function related indicators were measured before and after treatment, along with right-to-left shunting status, and incidence of complications in both groups. Results: There was no significant difference in the duration of surgery or hospitalization between the TTE group and the X-ray group (p>0.05). After treatment, the cardiac function indicators of both groups increased compared to before treatment (p<0.05), and there was no significant difference between the groups (p>0.05). After treatment, right-to-left shunting in the two groups improved compared to before treatment (p<0.05), with no significant difference between the groups (p>0.05). There was no significant difference in complications between the two groups (p>0.05). Conclusions: TTE guided PFO occlusion is as effective as X-ray guided PFO occlusion in the treatment of PFO. TTE surgery is clinically beneficial for reducing radiation damage with a good safety profile.

The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF)-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells.

Glial cell line-derived neurotrophic factor (GDNF), a potential therapeutic factor for Parkinson's disease (PD), exerts its biological effects through the Ret receptor tyrosine kinase. The redistribution of Ret into lipid rafts substantially influences Ret signaling, but the mechanisms underlying Ret translocation remain unclear. The purpose of our study was to further explore the signaling mechanisms of GDNF and to determine whether the actin cytoskeleton is involved in the GDNF-induced Ret translocation into lipid rafts. In MN9D dopaminergic neuronal cells, we used density gradient centrifugation and immunofluorescence confocal microscopy to separate and visualize lipid rafts, co-immunoprecipitation to analyze protein-protein interactions, and latrunculin B (Lat B) and jasplakinolide (Jas) to disrupt and enhance the polymerization of the actin cytoskeleton, respectively. The results showed that Ret translocated into lipid rafts and coimmunoprecipitated with actin in response to GDNF treatment. After Lat B or Jas treatment, the Ret-F-actin association induced by GDNF was impaired or enhanced respectively and then the levels of Ret translocated into lipid rafts were correspondingly inhibited or promoted. These data indicate that actin polymerization and cytoskeletal remodeling are integral to GDNF-induced cell signaling in dopaminergic cells and define a new role of the actin cytoskeleton in promoting Ret redistribution into lipid rafts.

Hypoxia pretreatment of bone marrow-derived mesenchymal stem cells seeded in a collagen-chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have properties that make them promising for the treatment of chronic nonhealing wounds. The major challenge is ensuring an efficient, safe, and painless delivery of BM-MSCs. Tissue-engineered skin substitutes have considerable benefits in skin damage resulting from chronic nonhealing wounds. Here, we have constructed a three-dimensional biomimetic scaffold known as collagen-chitosan sponge scaffolds (CCSS) using the cross-linking and freeze-drying method. Scanning electron microscopy images showed that CCSS had an interconnected network pore configuration about 100 µm and exhibited a suitable swelling ratio for maintaining morphological stability and appropriate biodegradability to improve biostability using swelling and degradation assays. Furthermore, BM-MSCs were seeded in CCSS using the two-step seeding method to construct tissue-engineered skin substitutes. In addition, in this three-dimensional biomimetic CCSS, BM-MSCs secreted their own collagen and maintain favorable survival ability and viability. Importantly, BM-MSCs exhibited a significant upregulated expression of proangiogenesis factors, including HIF-1α, VEGF, and PDGF following hypoxia pretreatment. In vivo, hypoxia pretreatment of the skin substitute observably accelerated wound closure via the reduction of inflammation and enhanced angiogenesis in diabetic rats with hindlimb ischemia. Thus, hypoxia pretreatment of the skin substitutes can serve as ideal bioengineering skin substitutes to promote optimal diabetic skin wound healing.

Surface Engineering of HEK293 Cell-Derived Extracellular Vesicles for Improved Pharmacokinetic Profile and Targeted Delivery of IL-12 for the Treatment of Hepatocellular Carcinoma.

Background: Extracellular vesicles (EVs) are considered a promising drug delivery platform. Naïve EVs face numerous issues that limit their applications, such as fast clearance, hepatic accumulations, and a lack of target-specific tropism. We aimed to explore a series of surface engineering approaches to: 1) reduce the non-specific adhesion of EVs, and 2) improve their enrichment in the target tissue. As a proof-of-concept, we investigated the therapeutic potentials of a multi-modal EVs system carrying a tumor-specific nanobody and the immuno-stimulant interleukin-12 (IL12) using in vivo models of hepatocellular carcinoma. Methods: The major cell adhesion molecule on the HEK293-derived EVs, integrin ß1 (ITGB1), was knocked out (KO) by CRISPR/Cas9-mediated gene editing, followed by deglycosylation to generate ITGB1-Deg EVs for the subsequent pharmacokinetic and biodistribution analyses. ITGB1-Deg EVs were further loaded with glypican-3 (GPC3)-specific nanobody (HN3) and mouse single-chain IL12 (mscIL12) to generate ITGB1-mscIL12+HN3+Deg EVs, for evaluation of tumor tropism and therapeutic potential in a mice model of hepatocellular carcinoma. Results: Removal of ITGB1 led to the broad suppression of integrins on the EVs surface, resulting in a decrease in cellular uptake. Deglycosylation of ITGB1- EVs gave rise to inhibition of the EVs uptake by activated RAW264.7 cells. ITGB1 removal did not significantly alter the pharmacokinetic behaviors of HEK293-EVs, whereas the ITGB1-Deg EVs exhibited enhanced systemic exposure with reduced hepatic accumulation. Loading of HN3 conferred the ITGB1-Deg EVs with tumor-specific tropism for both subcutaneous and metastasized tumors in mice. The ITGB1-mscIL12+HN3+Deg EVs activated mouse splenocytes with high potency. Systemic administration of the EVs with the equivalent dose of 1.5µg/kg of exosomal IL12 achieved satisfactory tumor growth inhibition and good tolerability. Conclusion: The combinatorial approach of EVs surface engineering conferred HEK293-EVs with reduced non-specific clearance and enhanced tumor targeting efficacy, which constituted an efficient delivery platform for critical cancer therapeutics like IL12.

Liraglutide-Loaded Milk Exosomes Lower Blood Glucose When Given by Sublingual Route.

Bovine milk is rich in extracellular vesicles (mEVs) which have been suggested as a possible drug delivery vehicle with oral bioavailability. As the digestive fluids contain many lipid- and protein-degrading enzymes, we explored whether mEVs given sublingually could be taken up systemically. mEVs were isolated using three different protocols, which were 120 nm in diameter and carried bovine CD81. Fluorescently stained mEVs given by sublingual route were detected in the circulation, whereas mEVs given by gavage were detected at 2-Log lower concentrations. As proof of the concept, we loaded mEVs with the antidiabetic drug Liraglutide (LRT-EV), which reduced blood glucose levels when given by the sublingual route but showed no efficacy via gavage. This study suggests that mEV may be an efficient delivery vehicle for drugs that are not orally bioavailable, and LRT-loaded EVs have the potential as the next-generation drug delivery platform for the treatment of chronic diseases, including diabetes.

Stable expression of a truncated TLX variant drives differentiation of induced pluripotent stem cells into self-renewing neural stem cells for production of extracellular vesicles.

BACKGROUND: Neural stem cells (NSCs)-derived extracellular vesicles (EVs) possess great potential in treating severe neurological and cerebrovascular diseases, as they carry the modulatory and regenerative ingredients of NSCs. Induced pluripotent stem cells (iPSCs)-derived NSCs culture represents a sustainable source of therapeutic EVs. However, there exist two major challenges in obtaining a scalable culture of NSCs for high-efficiency EVs production: (1) the heterogeneity of iPSC-derived NSCs culture impairs the production of high-quality EVs and (2) the intrinsic propensity of neuronal or astroglial differentiation of NSCs during prolonged culturing reduces the number of NSCs for preparing EVs. A NSCs strain that is amenable to stable self-renewal and proliferation is thus greatly needed for scalable and long-term culture. METHODS: Various constructs of the genes encoding the orphan nuclear receptor NR2E1 (TLX) were stably transfected in iPSCs, which were subsequently cultured in a variety of differentiation media for generation of iNSCsTLX. Transcriptomic and biomarker profile of iNSCsTLX were investigated. In particular, the positivity ratios of Sox2/Nestin and Musashi/Vimentin were used to gauge the homogeneity of the iNSCsTLX culture. The iNSCs expressing a truncated version of TLX (TLX-TP) was expanded for up to 45 passages, after which its neuronal differentiation potential and EV activity were evaluated. RESULTS: Stable expression of TLX-TP could confer the iPSCs with rapid and self-driven differentiation into NSCs through stable passaging up to 225 days. The long-term culture of NSCs maintained the highly homogenous expression of NSC-specific biomarkers and potential of neuronal differentiation. EVs harvested from the TLX-expressing NSCs cultures exhibited anti-inflammatory and neuroprotective activities. CONCLUSIONS: iPSC-derived NSCs stably expressing TLX-TP is a promising cell line for scalable production of EVs, which should be further exploited for therapeutic development in neurological treatment.

[The clinical characteristics and treatment of community outbreaks of the new influenza A (H1N1) infection].

OBJECTIVE: To describe the clinical characteristics of 3 community outbreaks of the novel influenza A (H1N1), and to compare the treatment effects of the traditional Chinese medicine with or without Oseltamivir. METHOD: The clinical records of 234 patients in 3 community outbreaks of the novel influenza A (H1N1) infection in June (n = 56), August (n = 96) and October (n = 82) of 2009 were analyzed, and the treatment effects of the traditional Chinese medicine with or without Oseltamivir were evaluated. RESULTS: The baseline characteristics, including age, temperature, indices of blood tests, hepatic and renal functions were distributed evenly between the 2 treatment groups. The overall analysis suggested that there was no significant difference between the 2 treatment groups in the duration of clinical symptoms (P > 0.05), the duration of fever (P > 0.05), and the hospitalization days (P > 0.05). However, an analysis stratified by the temperature (≥ 39°C or < 39°C) suggested that patients treated by the traditional Chinese medicine with Oseltamivir tended to suffer a shorter duration of fever [40.5 (37.3, 42.0) vs 22.0 (10.5, 30.8) hr, P < 0.01) ] in the higher temperature group. CONCLUSIONS: The traditional Chinese medicine was equivalent to oseltamivir in treating patients with the novel influenza A (H1N1) infection with lower temperature (< 39°C). Oseltamivir was effective in shortening the duration of fever in patients with temperature higher than 39°C.

[Clinical investigation on the compliance and the validity of ventilator bundle].

OBJECTIVE: To investigate the compliance of ventilator bundle implementation and its preventive effect on ventilator associated pneumonia (VAP). METHODS: A before and after design was used in this single center study. Patients aged from 18 to 80 years, with mechanical ventilation (MV) duration over 48 hours were recruited during 1 year before (control group) and 2 years after bundle implementation (intervention group). Measurements included the rate of successful ventilator bundle implementation in intervention group, incidence of VAP, duration of MV and mortality within 28 days in both groups. RESULTS: A total number of 237 patients, including 71 patients in control arm and 166 patients in intervention arm, were recruited in this study. There was no statistical significance in ratio of sex, mean age, category of diseases or mean acute physiology and chronic health evaluation II (APACHE II) score between two groups (all P>0.05). Significant changes were not found in MV duration [(5.9+/-5.6) days vs. (5.2+/-6.1) days], incidence of VAP (21.1% vs. 20.5%) and mortality within 28 days (16.9% vs. 19.8%) between control and intervention group as well. In intervention group, 57 of 166 (34.3%) patients were successfully implemented all of four ventilator bundle items. The successful rate of ventilator bundle implementation were 62.5% (35/56), 22.1% (21/95) and 6.7% (1/15) in patients received MV duration < or =3 days, 4-7 days and >7 days respectively. Among the four items of the bundle, head of bed elevation > or =30 degree angle had the lowest successful rate [43.4% (72/166)]. But it was much better in the implementation of daily wake-up plus weaning, prevention of peptic ulcer and prevention of deep vein thrombosis formation [92.2% (153/166), 88.0% (146/166) and 83.1% (138/166) respectively]. CONCLUSION: The poor compliance of ventilator bundle is an important factor in impacting the efficacy of ventilator bundle.

Infusion of adipose­derived mesenchymal stem cells inhibits skeletal muscle mitsugumin 53 elevation and thereby alleviates insulin resistance in type 2 diabetic rats.

It is widely accepted that infusion of mesenchymal stem cells (MSCs) ameliorates hyperglycemia by alleviating insulin resistance in rats with type 2 diabetes mellitus (T2D). However, the detailed underlying mechanisms are not clearly defined. Mitsugumin 53 (MG53) is an E3 ligase that has recently been implicated in the aggravation of insulin resistance by promoting the ubiquitinoylation of insulin receptor substrate­1 (IRS­1) in skeletal muscles. It was therefore hypothesized that MG53 may be involved in MSC­mediated therapeutic effects on insulin resistance. To test this hypothesis, in the present study, T2D rat models were induced by a high­fat diet combined with streptozotocin administration and MSC infusion was performed four times (once every 2 weeks for 8 weeks). The therapeutic effects of MSC infusion on insulin resistance were evaluated and the effect on the expression of MG53 and insulin receptor signaling elements in skeletal muscle was also investigated by immunofluorescence staining and western blotting. The results demonstrated that MSC infusion ameliorated hyperglycemia and insulin resistance in T2D rats. Furthermore, MSC infusion inhibited MG53 elevation and reversed the decreases in glucose transporter type 4, insulin receptor, IRS­1 and phosphorylated­AKT levels in the skeletal muscle of T2D rats. These results indicated that MSC infusion has therapeutic effects in rats and that MG53 in skeletal muscle may be a promising novel therapeutic target protein for MSC­mediated amelioration of insulin resistance in T2D.

Let-7d microRNA Attenuates 6-OHDA-Induced Injury by Targeting Caspase-3 in MN9D Cells.

Parkinson's disease (PD) mainly results from the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), and the exact underlying mechanisms of the loss of DA neurons in PD remains largely unclear. The results of our previous work showed that let-7d was significantly downregulated in a 6-OHDA-induced cellular model of PD. However, the exact effect of let-7d on DA neural cells was unclear. In MN9D dopaminergic neuronal cells, we used a let-7d mimic and inhibitor to upregulate and downregulate the expression of let-7d, respectively, a cell counting kit to assess cell viability, and a TUNEL staining assay and flow cytometry to examine the cell death rate, and we found that let-7d could negatively regulate 6-OHDA-induced cell injury. Then, we verified that caspase-3 was a target gene of let-7d by using a dual-luciferase reporter system. Furthermore, using caspase-3 siRNA and a caspase-3-overexpression vector (without the 3'UTR) to respectively inhibit and increase the expression of caspase-3, we found that caspase-3 siRNA could reverse the cell injury induced by the let-7d inhibitor and that caspase-3 overexpression could reverse the protective effects of the let-7d mimic on 6-OHDA-induced cell injury. Taken together, these findings strongly suggest that let-7d plays an important role in DA neuronal cell injury and that the effects of let-7d are, at least in part, via the suppression of caspase-3 expression.

Spleen-Derived Anti-Inflammatory Cytokine IL-10 Stimulated by Adipose Tissue-Derived Stem Cells Protects Against Type 2 Diabetes.

Considering that the spleen plays an important role in the occurrence and development of diabetes, we aimed at investigating the role of the spleen in the treatment of type 2 diabetes (T2D) with adipose tissue-derived stem cells (ADSCs). We established a T2D/splenectomy (SPX) rat model by using high-fat diet/streptozotocin administration with SPX, assessed the therapeutic effects of ADSCs, and explored the possible mechanism. A single ADSC infusion was found to ameliorate hyperglycemia and insulin resistance in diabetic rats, accompanied by a considerable number of ADSCs homing to the spleens in T2D rats. Moreover, four times of infusion of ADSCs resulted in a more significant reduction of blood glucose and insulin resistance, whereas SPX exacerbated hyperglycemia and insulin resistance and attenuated the effects of ADSCs. In addition, ADSC infusion promoted anti-inflammatory cytokine interleukin (IL)-10 expression and inhibited pro-inflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor (TNF)-α expression in both the spleen and serum of T2D rats without SPX. ADSCs also inhibited serum IL-6, IL-1ß, and TNF-α expression, but cannot promote IL-10 expression in T2D rats with SPX. Therefore, these data indicate that the effect of ADSCs ameliorating hyperglycemia and insulin resistance may be partially through promoting spleen-derived anti-inflammatory cytokine IL-10 expression. These novel findings further confirmed the essential role of the spleen in the ADSC treatment of T2D and provide an important theoretical basis for the potential application of ADSCs in T2D therapy.

Preferred M2 Polarization by ASC-Based Hydrogel Accelerated Angiogenesis and Myogenesis in Volumetric Muscle Loss Rats.

Volumetric muscle loss (VML) injury resulted from massive muscle defects and diseases for which there are still no effective therapeutic treatments. This study aimed to investigate the effects of rat adipose-derived mesenchymal stem cells (rASCs) and rASCs-conditioned medium- (CM-) based type I collagen hydrogel on macrophage (MP) transition, myogenesis, and vascularization in the rat VML model. Laser Doppler results demonstrated much higher blood flow in the rASC- and CM-based hydrogel groups. qRT-PCR, hematoxylin and eosin, immunofluorescence, and Sirius Red staining manifested that both rASCs and CM-based hydrogel implantation accelerated muscle repair with upregulated angiogenesis and myogenesis, attenuated inflammation while facilitating M2 transition, and decreased the collagen deposition compared with the hydrogel group. In vitro experiments indicated that factors secreted from polarized M2 MPs could accelerate the migration and tube formation capacities of HUVECs. These results suggested that rASCs exerted immunomodulatory effects on MPs which further enhanced the proangiogenic potential on ECs to promote myogenesis and angiogenesis during muscle repair. These fundamental results support further clinical applications of ASCs for muscle loss injury.

A highly efficient chemoenzymatic approach toward glycoprotein synthesis.

[reaction: see text] A highly efficient endoglycosidase-catalyzed synthesis of homogeneous glycoproteins is described. By using ribonuclease B as a model system, it was demonstrated that Endo-A could efficiently attach a preassembled oligosaccharide to a GlcNAc-containing protein in a regio- and stereospecific manner, when the corresponding sugar oxazoline was used as the donor substrate. The method allows the synthesis of both natural and tailor-made N-linked glycoproteins in excellent yield.

G-CSF Administration after the Intraosseous Infusion of Hypertonic Hydroxyethyl Starches Accelerating Wound Healing Combined with Hemorrhagic Shock.

OBJECTIVE: To evaluate the therapeutic effects of G-CSF administration after intraosseous (IO) resuscitation in hemorrhagic shock (HS) combined with cutaneous injury rats. METHODS: The rats were randomly divided into four groups: (1) HS with resuscitation (blank), (2) HS with resuscitation + G-CSF (G-CSF, 200 µg/kg body weight, subcutaneous injection), (3) HS with resuscitation + normal saline solution injection (normal saline), and (4) HS + G-CSF injection without resuscitation (Unres/G-CSF). To estimate the treatment effects, the vital signs of alteration were first evaluated, and then wound closure rates and homing of MSCs and EPCs to the wound skins and vasculogenesis were measured. Besides, inflammation and vasculogenesis related mRNA expressions were also examined. RESULTS: IO infusion hypertonic hydroxyethyl starch (HHES) exhibited beneficial volume expansion roles and G-CSF administration accelerated wound healing 3 days ahead of other groups under hemorrhagic shock. Circulating and the homing of MSCs and EPCs at wound skins were significantly elevated at 6 h after G-CSF treatment. Inflammation was declined since 3 d while angiogenesis was more obvious in G-CSF treated group on day 9. CONCLUSIONS: These results suggested that the synergistical application of HHES and G-CSF has life-saving effects and is beneficial for improving wound healing in HS combined with cutaneous injury rats.

Resveratrol glucuronides as the metabolites of resveratrol in humans: characterization, synthesis, and anti-HIV activity.

Resveratrol is a natural product with diverse biological activities. We have previously reported that resveratrol possesses potent synergistic inhibitory activity against human immunodeficiency virus (HIV)-1 infection in combination with nucleoside analogs (Heredia et al. 2000. J Acquir Immune Defic Syndr 25:246-255). As a part of our program in developing resveratrol as a component for anti-HIV chemotherapy, we describe in this article the characterization, chemical synthesis, and biological effects of the human metabolites of resveratrol. We found that resveratrol was metabolized in humans into two metabolites, which were characterized as resveratrol-3-O- and 4'-O-glucuronides. For further biological studies, we reported two simple, alternative methods for the synthesis of the metabolites. The cytotoxic and antiviral activities of resveratrol and its metabolites were compared in cell culture experiments using human peripheral blood mononuclear cells. Whereas resveratrol was cytotoxic at > or =30 microM, no cytotoxicity was observed for the metabolites at concentrations as high as 300 microM. However, resveratrol showed strong synergistic anti-HIV activity with didanosine at 10 microM, but no synergistic effects were observed for either of the metabolites at up to 300 microM. Nevertheless, the in vitro activity of the metabolites (resveratrol glucuronides) may not necessarily reflect their in vivo function, given the fact that the ubiquitously existing human beta-glucuronidase could convert the metabolites back to resveratrol locally or systematically in vivo. The present studies have implications for future development of resveratrol and/or its derivatives as a chemotherapeutic agent.

Factors associated with low adherence to head-of-bed elevation during mechanical ventilation in Chinese intensive care units.

BACKGROUND: Elevating the head of bed (HOB) 30° - 45° has been widely supported as a means of ventilator associated pneumonia (VAP) prevention. However, it was poorly adhered in clinical practice. This observational study aimed to investigate the factors impeding this simple practice at the bedside. METHODS: This prospective study was conducted in 33 Chinese academic hospital intensive care units (ICUs). HOB angle was measured four times daily at 5 - 7 hour intervals. The predefined HOB elevation goal was an angle ≥ 30°. RESULTS: The overall rate of achieving the HOB goal was 27.8% of the 8647 measurements in 314 patients during 2842 ventilation days. The HOB goal of ≥ 3 times/d was consistently achieved only in 15.9% of the cases. Almost 60% of patients had at least one 24 hours period during which the HOB goal was never documented. This low rate of protocol compliance was not associated with acute physiology and chronic health evaluation (APACHE) II score or dependence on vasopressors. In a survey, "nurse workload" was identified as the most important factor for non-compliance with the HOB goal. In addition, the rates of compliance were significantly different (P < 0.001) between physicians self-reporting that they either did or did not know the Institutes of Healthcare Improvement (IHI) ventilator bundle. CONCLUSIONS: Low adherence to a HOB angle of ≥ 30° was found in this nationwide survey. Nursing workload and lack of knowledge on VAP prevention were important barriers to changing this practice.

Synthesis and preliminary evaluation of n.c.a. iodoquine: a novel radiotracer with high uptake in cells with high ALDH1 expression.

PURPOSE: Chloroquine has demonstrated high affinity for aldehyde dehydrogenase 1A1 (ALDH1), an enzyme expressed in the highly tumorigenic CD133+ brain tumor initiating subpopulation. The purpose of this study is to report the novel synthesis of a chloroquine analogue, n.c.a. iodoquine, and the in vitro and in vivo uptake in cells with high ALDH1 content. METHODS AND MATERIALS: Iodoquine was synthesized in novel no-carrier-added forms (n.c.a.) for both 125I and 123I. I25I IQ and 18F FDG cell uptake assays were performed in the L1210 and L1210cpa (cyclophosphamide resistant), A549, and MG456 glioblastoma cell lines. Uptake was expressed as a percent of the administered activity. 125I IQ biodistribution studies assessed organ uptake at 1, 4, and 24 hours after IV administration (n= 15 total; 5 mice/timepoint). Radiation dosimetry estimates were calculated using standard OLINDA/EXM software. In vivo imaging of 123I IQ uptake in MG456 glioblastoma mouse model (n=10) was performed with small animal high resolution micro-SPECT. Autoradiography and histology co-localized radiotracer and tumor biodistribution. Uptake in MG456 glioblastoma tumors was quantified with gamma counting. RESULTS: L1210 cpa (high ALDH1) showed significantly higher 125I IQ uptake compared to the parental L1210 (low ALDH1) for all time points through 4 hours (20.7% ± 1.4% versus 11.0% ± 0.5%; 21.3% ± 0.9% versus 11.0% ± 0.4%; 20.6% ± 0.7% versus 9.4% ± 0.3%; and 15.7% ± 0.7% versus 7.5% + 0.4% at 30 minutes, and 1, 2 and 4 hours, respectively; p < 0.001 for all time points). In the CD133+ fraction of MG456 glioblastoma cell line, IQ uptake was significantly higher compared to FDG at all time points through 4 hours (81.5% ± 0.9% versus 1.3% ± 0.1%; 88.8% ± 0.4% versus 1.3% ± 0.1%; 87.8% ± 2.1% versus 1.7% ± 0.2%; and 87.0% ± 2.4% versus 1.8% ± 0.1 at 30 minutes, and 1, 2 and 4 hours, respectively; p > 0.001 for all time points). The A549 lung cancer cell line also showed high IQ uptake through 4 hours. IQ normal biodistribution studies showed rapid renal excretion and very low normal background brain activity after IV administration. In vivo micro-SPECT images showed mild uptake in larger MG456 glioblastomas (n=6) as verified with autoradiography and histology. Gamma well counter uptake in large tumors was 2.3% ± 0.48% ID/g (n=5). CONCLUSION: Iodoquine localizes to cells with high ALDH1 content. Cell assays show high 125I IQ uptake in the MG456 cell line, and in vivo micro-SPECT imaging showed mild 123I IQ uptake in MG456 glioblastomas. Further studies are necessary to investigate 131I IQ as a potential therapeutic agent targeting the highly tumorigenic CD133+ brain tumor stem cell subpopulation.

[Construction of eukaryotic plasmid of human GPC3 and expression and purification of recombinant GPC3 protein].

AIM: To obtain enough human glypican-3 (GPC3) protein for structural and functional research. METHODS: The full-length cDNA coding for GPC3 was cloned by RT-PCR from human fetal hepatocytes. The open reading frame (ORF) of the cDNA consists of 1 700 bases, encoding a mature protein of 556 amino acids. The cDNA was inserted into the pPICZ A vector to construct a expression plasmid, named pPICZ A-GPC3. Then the plasmid was transformed into a Pichia pastoris strain, GS115 and the positive strains were screened on the YPD plates with Zeocin. The positive strains were further screened on cellulose acetate and nitrocellulose membrane with HRP labeled His-tag antibody. The selected strains were induced by methanol and the supernatants were analyzed by SDS-PAGE and Western blotting. RESULTS: SDS-PAGE analysis showed an anticipated band on the gel that could bind with goatanti-GPC3 antibody. Furthermore, the strain was fermented and the expression level was about 5 mg/L, and the recombinant GPC3 protein was purified by cation-exchange chromatography from the fermentation supernatant. CONCLUSION: Human GPC3 was expressed successfully in Pichia pastoris and purified to obtain the recombinant protein from fermentation supernatant, which made it possible for further structural and functional studies on GPC3.

Targeting aldehyde dehydrogenase: a potential approach for cell labeling.

INTRODUCTION: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. METHODS: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. RESULTS: The average radiochemical yields for the synthesis of [(125)I]FMIC and [(125)I]DEIBA were 70+/-5% and 47+/-14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. CONCLUSION: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

Toward a carbohydrate-based HIV-1 vaccine: synthesis and immunological studies of oligomannose-containing glycoconjugates.

Human antibody 2G12 is a broadly neutralizing antibody that exerts its anti-HIV activity by targeting a novel oligomannose cluster on HIV-1 gp120. It was previously demonstrated that synthetic oligomannose clusters could mimic the carbohydrate epitope of 2G12 and showed enhanced antigenicity (Wang L. X. et al. (2004) Chem.Biol. 11, 127). This paper describes the synthesis of oligomannose-containing glycoconjugates that include either a carrier protein or a universal T-helper epitope peptide to provide an effective immunogen. It was shown that the synthetic neoglycoconjugates containing oligomannose clusters could be recognized by the human antibody 2G12. Rabbit immunization studies revealed that only a small fraction of antibodies raised by the glycoconjugates was directed to the carbohydrate antigens, with the majority of the IgG type antibodies being directed to the linkers in the conjugates. The anti-sera showed weak cross-reactivity to HIV-1 gp120.