Vitamin D Levels Assessment among the Neonates with and without Seizures: A Single Center Cross-Sectional Study.

Introduction: The impact that vitamin D (vit D) has on a variety of medical conditions like diabetes, cardiovascular, oncological, and central nervous system disorders has been a topic of interest for many years now. It is well-known that vit D deficiency is substantially more common in epileptics than in healthy subjects. The current research was piloted to analyse the vit D levels of the blood in newborns with seizures, as well as mothers' vit D status included subjects. Materials and Methods: A cross-sectional examination was piloted at a tertiary care center, which had a neonatal intensive care unit (NICU). The subjects were neonates and their mothers. The levels of vit D were measured in term and late preterm newborns who had been brought to the NICU with convulsions. Term or late preterm infants who were healthy and hospitalized in the same hospital's postnatal unit as their mothers served as the controls for the study. Demographics, as well as the vit D levels of both the neonate and the mother, were estimated and compared and evaluated for any significance, keeping significance at less than 0.05. Results: Of the 72 neonates included, they were similarly distributed between the epileptic (37) and healthy subjects. (40) The mothersy subjects.cluded, they were sim D levels averaged 15.11 ded, they were similarly distributed b D levels of their newborns were 13.26 ± 5.12 ng/mL (P = 0.77). There was no significant variance between the healthy and epileptic neonates (P = 0.212). Conclusion: The current studyficant variance between the healthy and epileptic neonates (eptic with convulsions. Termserum vit D levels and epileptic activity in neonates. Nevertheless, the levels of the vitamin were < 20 ng/mL among all the neonates. Interventions to improve the vit D levels have to be implemented.

Improving the Knowledge and Competency of UK Foundation Doctors in Nasogastric Tube Placement: A National Study.

Objective This study aims to improve foundation doctors' knowledge of guidelines for confirming nasogastric (NG) tube position and to enhance their confidence and competency in NG tube placement. Methods A three-part educational approach was designed, which included an educational leaflet and allowed the assessment of a participant's knowledge of guidelines pertaining to NG tube positioning before and after education. This educational leaflet and accompanying pre- and post-learning assessments were distributed among NHS Foundation Trusts in the UK between January 2022 and June 2022. All participants were foundation doctors in the UK. Those who had entered further training after the completion of their foundation training, at the time of assessment distribution, were excluded. Results A total of 173 participants completed this assessment. We found a significant increase in confidence among participants following the education (p<0.05). There was also a significant improvement in objective knowledge of guidelines on NG tube position confirmation following education (p<0.05). Conclusions Current knowledge on NG tube positioning is lacking among foundation doctors, but this can be significantly improved with simple educational leaflets. Furthermore, many participants felt that more training is needed, and this topic should be included in an essential teaching program.

Plasmid-Mediated Antibiotic Resistant Escherichia coli in Sarawak Rivers and Aquaculture Farms, Northwest of Borneo.

BACKGROUND: The emergence of plasmid-mediated antibiotic resistance in Escherichia coli in water resources could pose a serious threat to public health. The study aims to investigate the dispersion of plasmid-mediated antibiotic-resistant E. coli from six rivers in Sarawak and two aquaculture farms in Borneo. METHODS: A total of 74 water samples were collected for the determination of their bacteria colony count. An IMViC test identified 31 E. coli isolates and tested their susceptibility against twelve clinically important antibiotics. The extraction of plasmid DNA was done using alkali lysis SDS procedures. Characteristics, including plasmid copy number, molecular weight size, resistance rate and multiple antibiotic resistance (MAR), were assessed. RESULTS: Our findings revealed that bacterial counts in rivers and aquaculture farms ranged from log 2.00 to 3.68 CFU/mL and log 1.70 to 5.48 cfu/mL, respectively. Resistance to piperacillin (100%) was observed in all E. coli; resistance to amoxicillin (100%) and ampicillin (100%) was observed in E. coli found in aquaculture farms; resistance to streptomycin (93%) was observed in E. coli found in rivers. All E. coli were resistant to ≥2 antibiotics and formed 26 MAR profiles, ranging from an index of 0.17 to 0.83, indicating that there are high risks of contamination. Some (48.4%) of the E. coli were detected with plasmids (1.2 to >10 kb), whereas 51.6% of the E. coli did not harbor any plasmids. The plasmid copy numbers reported were one plasmid (n = 7), two plasmids (n = 4), ≥ two plasmids (4). E. coli isolated from the Muara Tuang River showed the highest-molecular-weight plasmids. A statistical analysis revealed that there is no significant correlation (r = 0.21, p = 0.253) between the number of plasmids and the MAR index of the tested isolates. CONCLUSION: The distribution of MAR in E. coli from rivers is higher compared to the aquaculture environment. Our study suggests that MAR in isolates could be chromosome-mediated. Our results suggest that riverbed sediments could serve as reservoirs for MAR bacteria, including pathogens, under different climatic conditions, and their analysis could provide information for public health concerns.

Relationship between temporomandibular joint pain and magnetic resonance imaging findings in patients with temporomandibular joint disorders.

The purpose of this study was to evaluate abnormal magnetic resonance imaging (MRI) findings related to temporomandibular joint (TMJ) pain. This study included 245 joints of 152 patients with temporomandibular disorders with anterior disc displacement; of these, 129 joints had joint pain whereas 116 joints had no joint pain. MRI was used to evaluate the reduction of anterior disc displacement, joint effusion, mandible condylar morphology, bone marrow oedema of the mandibular condyle, and signal intensity of the posterior disc attachment (PDA) on fat-suppressed T2-weighted images. The odds ratio (OR) for each MRI variable for the pain group versus the no pain group was computed using logistic regression analysis. Univariate logistic regression analysis showed significant correlations between TMJ pain and all MRI findings. Multivariate logistic regression analysis showed significant correlations with joint effusion (P=0.03, OR 2.21), bone marrow oedema (P<0.001, OR 11.75), and signal intensity of the PDA (P<0.001, OR 6.21). These results suggest that bone marrow oedema, high signal intensity of the PDA on fat-suppressed T2-weighted images, and joint effusion, in descending order of influence, are factors related to TMJ pain.

The role of reflectance confocal microscopy in the diagnosis and management of squamous cell carcinoma in situ treated with photodynamic therapy.

BACKGROUND: Reflectance confocal microscopy (RCM) is increasingly used for noninvasive in vivo diagnosis of skin cancers. We seek to determine if RCM is useful for the diagnosis and follow-up of squamous cell carcinoma in situ (SCCIS) posttreatment to document clearance. METHODS: A pilot prospective study enrolled 10 patients with a total of 11 SCCIS lesions. Clinical, confocal, histological features and fluorescence diagnosis (FD) were recorded pre- and posttreatment. RESULTS: Four SCCIS lesions underwent RCM imaging prior to biopsy, while 11 SCCIS lesions were followed up with RCM imaging. Clinical features of persistent SCCIS post-PDT in four out of 11 follow-up cases were confirmed with RCM and FD. There were no RCM features of SCCIS in seven lesions which were clinically cured. All eight (four new SCCIS and four follow-up) cases displayed atypical honeycomb pattern. Two cases (25%) showed numerous epidermal dendritic cells, while small bright refractive cells were present in the epidermis in two lesions (25%). Round blood vessels in the superficial dermis were seen in four lesions (50%), while three lesions (37.5%) showed dermal inflammatory cells. CONCLUSION: There was good correlation between histological and confocal features in patients who underwent RCM imaging prior to biopsy. RCM may be a complementary tool in diagnosing SCCIS and to monitor response to nonsurgical treatment by avoiding unnecessary biopsies especially in lesions with persistent residual postinflammatory erythema.

Periprosthetic UHMWPE Wear Debris Induces Inflammation, Vascularization, and Innervation After Total Disc Replacement in the Lumbar Spine.

BACKGROUND: The pathophysiology and mechanisms driving the generation of unintended pain after total disc replacement (TDR) remain unexplored. Ultrahigh-molecular-weight polyethylene (UHMWPE) wear debris from TDRs is known to induce inflammation, which may result in pain. QUESTIONS/PURPOSES: The purpose of this study was to determine whether (1) periprosthetic UHMWPE wear debris induces immune responses that lead to the production of tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß, the vascularization factors, vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGFbb), and the innervation/pain factors, nerve growth factor (NGF) and substance P; (2) the number of macrophages is associated with the production of the aforementioned factors; (3) the wear debris-induced inflammatory pathogenesis involves an increase in vascularization and associated innervation. METHODS: Periprosthetic tissues from our collection of 11 patients with contemporary TDRs were evaluated using polarized light microscopy to quantify UHMWPE wear particles. The major reason for revision (mean implantation time of 3 years [range, 1-6 years]) was pain. For control subjects, biopsy samples from four patients with degenerative disc disease with severe pain and autopsy samples from three normal patients with no history of back pain were also investigated. Immunohistochemistry and histology were used to identify secretory factors, macrophages, and blood vessels. Immunostained serial sections were imaged at ×200 magnification and using MATLAB and NIH ImageJ, a threshold was determined for each factor and used to quantify positive staining normalized to tissue sectional area. The Mann-Whitney U test was used to compare results from different patient groups, whereas the Spearman Rho test was used to determine correlations. Significance was based on p < 0.05. RESULTS: The mean percent area of all six inflammatory, vascularization, and innervation factors was higher in TDR tissues when compared with normal disc tissues. Based on nonparametric data analysis, those factors showing the most significant increase included TNFα (5.17 ± 1.76 versus 0.05 ± 0.03, p = 0.02), VEGF (3.02 ± 1.01 versus 0.02 ± 0.002, p = 0.02), and substance P (4.15 ± 1.01 versus 0.08 ± 0.04, p = 0.02). The mean percent area for IL-1ß (2.41 ± 0.66 versus 0.13 ± 0.13, p = 0.01), VEGF (3.02 ± 1.01 versus 0.34 ± 0.29, p = 0.04), and substance P (4.15 ± 1.01 versus 1.05 ± 0.46, p = 0.01) was also higher in TDR tissues when compared with disc tissues from patients with painful degenerative disc disease. Five of the factors, TNFα, IL-1ß, VEGF, NGF, and substance P, strongly correlated with the number of wear particles, macrophages, and blood vessels. The most notable correlations included TNFα with wear particles (p < 0.001, ρ = 0.63), VEGF with macrophages (p = 0.001, ρ = 0.71), and NGF with blood vessels (p < 0.001, ρ = 0.70). Of particular significance, the expression of PDGFbb, NGF, and substance P was predominantly localized to blood vessels/nerve fibers. CONCLUSIONS: These findings indicate wear debris-induced inflammatory reactions can be linked to enhanced vascularization and associated innervation/pain factor production at periprosthetic sites around TDRs. Elucidating the pathogenesis of inflammatory particle disease will provide information needed to identify potential therapeutic targets and treatment strategies to mitigate pain and potentially avoid revision surgery. LEVEL OF EVIDENCE: Level III, therapeutic study.

UHMWPE wear debris and tissue reactions are reduced for contemporary designs of lumbar total disc replacements.

BACKGROUND: Lumbar total disc replacement (L-TDR) is a procedure used to relieve back pain and maintain mobility. Contemporary metal-on-polyethylene (MoP) L-TDRs were developed to address wear performance concerns about historical designs, but wear debris generation and periprosthetic tissue reactions for these newer implants have not been determined. QUESTIONS/PURPOSES: The purpose of this study was to determine (1) whether periprosthetic ultrahigh-molecular-weight polyethylene (UHMWPE) wear debris and biological responses were present in tissues from revised contemporary MoP L-TDRs that contain conventional cores fabricated from γ-inert-sterilized UHMWPE; (2) how fixed- versus mobile-bearing design affected UHMWPE wear particle number, shape, and size; and (3) how these wear particle characteristics compare with historical MoP L-TDRs that contain cores fabricated from γ-air-sterilized UHMWPE. METHODS: We evaluated periprosthetic tissues from 11 patients who received eight fixed-bearing ProDisc-L and four mobile-bearing CHARITÉ contemporary L-TDRs with a mean implantation time of 4.1 and 2.7 years, respectively. Histologic analysis of tissues was performed to assess biological responses and polarized light microscopy was used to quantify number and size/shape characteristics of UHMWPE wear particles from the fixed- and mobile-bearing devices. Comparisons were made to previously reported particle data for historical L-TDRs. RESULTS: Five of seven (71%) fixed-bearing and one of four mobile-bearing L-TDR patient tissues contained at least 4 particles/mm(2) wear with associated macrophage infiltration. Tissues with wear debris were highly vascularized, whereas those without debris were more necrotic. Given the samples available, the tissue around mobile-bearing L-TDR was observed to contain 87% more, 11% rounder, and 11% less-elongated wear debris compared with tissues around fixed-bearing devices; however, there were no significant differences. Compared with historical L-TDRs, UHMWPE particle number and circularity for contemporary L-TDRs were 99% less (p = 0.003) and 50% rounder (p = 0.003). CONCLUSIONS: In this preliminary study, short-term results suggest there was no significant influence of fixed- or mobile-bearing designs on wear particle characteristics of contemporary L-TDRs, but conventional UHMWPE has notably improved the wear resistance of these devices compared with historical UHMWPE.

Rare complications of osteolysis and periprosthetic tissue reactions after hybrid and non-hybrid total disc replacement.

PURPOSE: Few complications have been reported for lumbar total disc replacement (TDR) and hybrid TDR fixations. This study evaluated retrieved implants and periprosthetic tissue reactions for two cases of osteolysis following disc arthroplasty with ProDisc-L prostheses. METHODS: Implants were examined for wear and surface damage, and tissues for inflammation, polyethylene wear debris (polarized light microscopy) and metal debris (energy-dispersive X-ray spectroscopy). RESULTS: Despite initial good surgical outcomes, osteolytic cysts were noted in both patients at vertebrae adjacent to the implants. For the hybrid TDR case, heterotopic ossification and tissue necrosis due to wear-induced inflammation were observed. In contrast, the non-hybrid implant showed signs of abrasion and impingement, and inflammation was observed in tissue regions with metal and polyethylene wear debris. CONCLUSIONS: In both cases, wear debris and inflammation may have contributed to osteolysis. Surgeons using ProDisc prostheses should be aware of these rare complications.

Which design and biomaterial factors affect clinical wear performance of total disc replacements? A systematic review.

BACKGROUND: Total disc replacement was clinically introduced to reduce pain and preserve segmental motion of the lumbar and cervical spine. Previous case studies have reported on the wear and adverse local tissue reactions around artificial prostheses, but it is unclear how design and biomaterials affect clinical outcomes. QUESTIONS/PURPOSES: Which design and material factors are associated with differences in clinical wear performance (implant wear and periprosthetic tissue response) of (1) lumbar and (2) cervical total disc replacements? METHODS: We performed a systematic review on the topics of implant wear and periprosthetic tissue response using an advanced search in MEDLINE and Scopus electronic databases. Of the 340 references identified, 33 were retrieved for full-text evaluation, from which 16 papers met the inclusion criteria (12 on lumbar disc replacement and five on cervical disc replacement; one of the included studies reported on both lumbar and cervical disc replacement), which involved semiquantitative analysis of wear and adverse local tissue reactions along with a description of the device used. An additional three papers were located by searching bibliographies of key articles. There were seven case reports, three case series, two case-control studies, and seven analytical studies. The Methodological Index for Non-randomized Studies (MINORS) Scale was used to score case series and case-control studies, which yielded mean scores of 10.3 of 16 and 17.5 of 24, respectively. In general, the case series (three) and case-control (two) studies were of good quality. RESULTS: In lumbar regions, metal-on-polymer devices with mobile-bearing designs consistently generated small and large polymeric wear debris, triggering periprosthetic tissue activation of macrophages and giant cells, respectively. In the cervical regions, metal-on-polymer devices with fixed-bearing designs had similar outcomes. All metal-on-metal constructs tended to generate small metallic wear debris, which typically triggered an adaptive immune response of predominantly activated lymphocytes. There were no retrieval studies on one-piece prostheses. CONCLUSIONS: This review provides evidence that design and biomaterials affect the type of wear and inflammation. However, clinical study design, followup, and analytical techniques differ among investigations, preventing us from drawing firm conclusions about the relationship between implant design and wear performance for both cervical and lumbar total disc replacement.

In vitro metabolism of 3,4-methylenedioxymethamphetamine in human hepatocytes.

Users of the illicit drug, 3,4-methylenedioxymethamphetamine (MDMA), show signs of neurotoxicity. However, the precise mechanism of neurotoxicity caused by use of MDMA has not yet been elucidated. Synthetic glutathione (GSH) conjugates of MDMA are transported into the brain by the GSH transporter and subsequently produce neurotoxicity. The objective of this research is to show direct evidence of the formation of GSH adducts of MDMA in human hepatocytes. High-performance liquid chromatography coupled with tandem mass spectrometry was utilized to examine in vitro incubations of MDMA with cryopreserved human hepatocytes. The use of hydrophilic liquid chromatography in combination with linear ion trap mass spectrometry permitted the identification of two possible GSH metabolites. Enhanced product ion scans of m/z = 499 and 487 amu of extracts from hepatocytes treated with 1.0 mM MDMA show a distinct fragmentation pattern (m/z 194.2, 163, 135, 105), suggesting the formation of MDMA-GSH conjugate, MDMA-SG and 3,4-dihydroxymethamphetamine-SG. The formation of an MDMA-GSH conjugate was further supported by the apparent lack of the same fragmentation pattern from hepatocyte samples without MDMA treatment. The results generated from this study yield valuable qualitative and quantitative information about the neurotoxic thioether metabolites formed from MDMA in humans.

Integrin α3 is overexpressed in glioma stem-like cells and promotes invasion.

BACKGROUND: Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs. METHODS: Neurospheres and CD133⁺ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines. RESULTS: Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⁺ cells compared with CD133⁻ cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. CONCLUSION: Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Elimination of teicoplanin by adsorption to the filter membrane during haemodiafiltration: screening experiments for linezolid, teicoplanin and vancomycin followed by in vitro haemodiafiltration models for teicoplanin.

Pharmaceutical agents directed against methicillin-resistant Staphylococcus aureus can be eliminated during haemodiafiltration, not only by diffusion and ultrafiltration, but also by adsorption onto haemofilters. The latter may be affected by the binding of agents to serum albumin. The present study therefore investigated the affinity of anti-methicillin-resistant Staphylococcus aureus agents (teicoplanin, linezolid, vancomycin) for haemofilters and the pharmacokinetic properties of teicoplanin during haemodiafiltration. Linezolid, teicoplanin and vancomycin were first screened for their in vitro affinity for three different kinds of filter membranes: polysulfone, polyacrylonitrile and polymethylmethacrylate. Only teicoplanin showed significant filter-binding activity. An in vitro haemodiafiltration circulation model was then developed that incorporated a one-litre beaker containing Krebs-Ringer's bicarbonate solution with/without human albumin (0 or 3 g/dl) as an artificial plasma. Teicoplanin (initial concentration 50 µg/ml, representing the maximum plasma concentration (Cmax) resulting from a typical clinical dosage) was circulated throughout the beaker. Teicoplanin concentrations in the 'plasma' and ultrafiltrate were determined by high performance liquid chromatography. In the screening experiment, teicoplanin was predominantly adsorbed onto polysulfone and polymethylmethacrylate membranes. Furthermore, teicoplanin was primarily eliminated by adsorption onto these filters during in vitro haemodiafiltration. Albumin significantly reduced both haemodiafiltration clearance and the adsorption-dependent elimination, although there were complex but significant interactions between albumin and the filter membrane. Elimination of teicoplanin in an in vitro haemodiafiltration model was largely due to adsorption onto polysulfone and polymethylmethacrylate haemofilters. Future clinical studies should likely be designed to evaluate present recommendations of teicoplanin dosages in patients on haemodiafiltration.

Roles of TauT and system A in cytoprotection of rat syncytiotrophoblast cell line exposed to hypertonic stress.

The purpose of this study was to clarify the cytoprotective mechanism(s) induced in a conditionally immortalized syncytiotrophoblast cell line (TR-TBT 18d-1) exposed to hypertonic conditions. Hypertonicity-induced apoptosis of TR-TBT 18d-1 cells, but this was blocked by addition of 1 mM taurine to the culture medium. TauT-knockdown using siRNA revealed that TauT is a major contributor to taurine uptake by TR-TBT 18d-1 cells, at least under normal conditions. Cellular uptake of [(3)H]taurine and [(14)C]betaine by TR-TBT 18d-1 cells cultured under hypertonic conditions was increased compared to that under normal conditions. TauT, BGT-1, ATA2 and HSP70 mRNAs were upregulated by hypertonicity, while OCTN2, ENT1 and CNT1 mRNAs were downregulated. [(3)H]Taurine uptake was strongly inhibited by TauT inhibitors such as hypotaurine and ß-alanine. MeAIB, a system A specific substrate, inhibited hypertonic stress-induced [(14)C]betaine uptake. These results suggest that TauT and system A play cytoprotective roles in syncytiotrophoblasts exposed to hypertonic stress.

Influx mechanism of 2',3'-dideoxyinosine and uridine at the blood-placenta barrier.

The blood-placenta barrier (BPB) serves to protect the fetus from exposure to toxins, and to transport various nutrients, including nucleosides, and hormones from mother to fetus. It is known that nucleoside transporters contribute to the transfer of nucleosides and nucleoside analogues. 2',3'-Dideoxyinosine (ddI) has a nucleoside structure, and crosses the BPB. Although ddI is a substrate of several transporters, including equilibrative nucleoside transporters (ENT1 and ENT2), the transport mechanism of ddI in the placenta has not yet been characterized. Therefore, the purpose of this study was to clarify the influx mechanisms of ddI from the maternal to the fetal side, and to examine the interaction between ddI and uridine transport at the BPB. We studied ddI and uridine uptakes using a conditionally immortalized rat syncytiotrophoblast cell line, TR-TBT 18d-1, as a BPB model. The ddI uptake was temperature-dependent, Na(+)-independent and saturable. Kinetic analysis yielded K(m) values for ddI and uridine of 6.51 mM and 23.4 microM, respectively. Uridine uptake was inhibited by ENT1 and ENT2 substrates, and ddI uptake was also inhibited by substrates or inhibitors at concentrations that inhibit ENT2. Uridine uptake in Xenopus laevis oocytes expressing rat ENT2 was inhibited by 5mM ddI, in agreement with the results for TR-TBT 18d-1. Our results indicate that ddI and uridine are both taken up in part via ENT2 in TR-TBT 18d-1 cells, and therefore that ENT2 may contribute to their uptake at the BPB.

Potential of various drugs to inhibit nucleoside uptake in rat syncytiotrophoblast cell line, TR-TBT 18d-1.

The placenta requires nucleosides as nutrients for fetal growth, so it is important to examine potential interactions between placental transports of nucleosides and drugs to ensure the safety of pharmacotherapy during pregnancy. The purposes of this study are to clarify the uptake mechanisms of nucleosides from the maternal side of the syncytiotrophoblast and to investigate the inhibitory effect of various drugs on nucleoside uptake, using the rat syncytiotrophoblast cell line TR-TBT 18d-1, which shows syncytial-like morphology and functional expression of several transporters. Initial uptake of [(3)H]uridine or [(3)H]adenosine from the apical side of TR-TBT 18d-1 was markedly reduced by an excess of the respective unlabelled compound, and was slightly reduced by replacement of Na(+) with N-methyl-d-glucamine, indicating that both uptakes were Na(+)-independent. [(3)H]Uridine and [(3)H]adenosine uptakes in the absence of Na(+) were significantly and concentration-dependently inhibited by both 0.1 microM and 100 microM nitrobenzylthioinosine, suggesting the involvement of equilibrative nucleoside transporters (ENTs, SLC29). Kinetic analysis of adenosine uptake yielded a K(m) value of approximately 17 microM. These results are consistent with the reported uptake characteristics of uridine and adenosine by ENT1 and ENT2. The uptakes were significantly reduced by high concentrations of several nucleoside drugs, including cytarabine, vidarabine, zidovudine, mizoribine, caffeine and amitriptyline, but the effects were small within the therapeutic concentration ranges. In summary, our results suggest that ENTs are involved in apical uptake of uridine and adenosine in the syncytiotrophoblast. However, therapeutic concentrations of the drugs tested in this study might have little influence on maternal-to-fetal nucleoside transfer.

Characterization of the mechanism of zidovudine uptake by rat conditionally immortalized syncytiotrophoblast cell line TR-TBT.

PURPOSE: To characterize the uptake mechanism of zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, in syncytiotrophoblast cells using the TR-TBT 18d-1 cell line previously established by our group. MATERIALS AND METHODS: The effects of several transporter inhibitors on the initial and steady-state apical uptake of AZT by TR-TBT 18d-1 were characterized, in order to identify the transporter(s) involved. RESULTS: Initial uptake of AZT was sodium-independent and saturable; the K(m) value was about 16 microM. Nitrobenzylthioinosine (NBMPR), probenecid and cimetidine each had little effect on the saturable AZT uptake, indicating that well characterized transporters, such as organic anion transporters (OATs and OATPs), organic cation transporters (OCTs) and equilibrative nucleoside transporters (ENTs), are not involved. However, thymidine and 2'-deoxyuridine strongly inhibited AZT uptake. These results suggest that an unidentified nucleoside uptake transporter is responsible for the uptake of AZT. Cyclosporin A, Ko143 and probenecid had little effect on AZT accumulation by TR-TBT 18d-1 cells, suggesting that transporter-mediated efflux of AZT is not substantial. CONCLUSION: Our results indicate that saturable AZT uptake into TR-TBT 18d-1 is mediated by a so-far-unidentified transporter.

Mesenchymal stem cell-based angiopoietin-1 gene therapy for acute lung injury induced by lipopolysaccharide in mice.

Bone marrow-derived mesenchymal stem cells (MSCs) can serve as a vehicle for gene therapy. Angiopoietin-1 (Ang1) is a critical factor for endothelial survival and vascular stabilization via the inhibition of endothelial permeability and leukocyte-endothelium interactions. We hypothesized that MSC-based Ang1 gene therapy might be a potential therapeutic approach for lipopolysaccharide (LPS)-induced lung injury. MSCs were isolated from 6 week-old inbred male mice and transduced with the Ang1 gene, using a lentivirus vector. The MSCs showed no significant phenotypic changes after transduction. In the in vivo mouse model, the LPS-induced lung injury was markedly alleviated in the group treated with MSCs carrying Ang1 (MSCs-Ang1), compared with groups treated with MSCs or Ang1 alone. The expression of Ang1 protein in the recipient lungs was increased after MSCs-Ang1 administration. The histopathological and biochemical indices of LPS-induced lung injury were improved after MSCs-based Ang1 gene treatment. MSCs-Ang1 administration also reduced pulmonary vascular endothelial permeability and the recruitment of inflammatory cells into the lung. Cells of MSC origin could be detected in the recipient lungs for 2 weeks after injection with MSCs. These results suggest that MSCs and Ang1 have a synergistic role in the treatment of LPS-induced lung injury. MSC-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of acute lung injury.

Hepatocyte growth factor and c-Met expression in pericytes: implications for atherosclerotic plaque development.

Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c-Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31-positive endothelial cells. c-Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT-PCR, we demonstrated expression of HGF and c-Met in a rat pericyte cell-line, TR-PCT1, and in primary pericytes. HGF treatment of TR-PCT1 cells induced their migration, but not their proliferation, in a dose-dependent manner (10-100 ng/ml, p<0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 microM) or LY294002 (10 microM) abolished these effects. This work demonstrates the expression of c-Met and HGF in human atherosclerotic arteries, in association with SM-actin-positive cells and CD-31-positive cells, respectively. HGF induces pericyte migration via PI3-kinase and Akt activation in vitro. HGF and c-Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage.