Transferrin (Tf) conjugated lipopolyplexes (LPs) carrying G3139, an antisense oligonucleotide for Bcl-2, were synthesized and evaluated in Tf receptor positive K562 erythroleukemia cells and then in a murine K562 xenograft model.
Materials and Methods
Particle size and Zeta potentials of transferrin conjugated lipopolyplexs containing G3139 (Tf-LP-G3139) were measured by Dynamic Light Scattering and ZetaPALS. In vitro and in vivo sample’s Bcl-2 downregulation was analyzed using Western blot and tumor tissue samples also exhibited by immunohistochemistry method. For athymic mice bearing with K562 xenograft tumors, tumor growth inhibition and survival rate were investigated. Nanoparticle distribution in 3-D cell cluster was observed by Laser scan confocal microscopy. IL-12 production in the plasma was measured by ELISA kit.
In vitro, Tf-LP-G3139 was more effective in inducing down regulation of Bcl-2 in K562 cells than non-targeted LP-G3139, free G3139 and mismatched control ODN-G4126 in the same formulation. In vivo Tf-LP-G3139 was less effective than free G3139 in Bcl-2 down regulation. 3-D cell cluster model diffusion results indeed indicated limited penetration of the LPs into the cell cluster. Finally, the therapeutic efficacies of Tf-LP-G3139 and free G3139 were determined in the K562 xenograft model. Tf-LP-G3139 showed slower plasma clearance, higher AUC, and greater accumulation in the tumor compared to free G3139. In addition, Tf-LP-G3139 was found to be more effective in tumor growth inhibition and prolonging mouse survival than free G3139. This was associated with increased spleen weight and IL-12 production in the plasma.
The role of the immune system in the therapeutic response obtained with the Tf-LPs is necessary and in vitro 3-D cell cluster model can be a potential tool to evaluate the nanoparticle distribution.
Bcl-2; leukemia; lipopolyplexs; targeted drug delivery; transferrin receptor
Oligonucleotides (ONs) have shown great promise as therapeutic agents for various diseases. It is necessary to provide a protocol for preparation of ON-loaded lipid nanoparticles (LNPs) in a reproducible manner on a laboratory scale.
Materials and Methods
A 3-inlet microfluidic (MF) chip-based device was used to synthesize LNPs at the lipid/ON ratio of 10/1 (w/w) and at flow rates ranging from 50 to 1100 µl/min. A series of LNPs containing either antisense oligodeoxyribonucleotide (AS-ODN) or small-interfering RNA (siRNA) were synthesized. Bulk mixing was used as control.
The MF method was shown to be particularly useful for synthesis of LNPs loaded with AS-ODN. The optimal range of flow rates for AS-ODN LNPs was found to be 100 to 200 µl/min. MF synthesis produced LNPs with lower polydispersity values. However, the MF was less effective in preparing LNPs loaded with siRNA, which may have been due to greater rigidity of double-stranded siRNA comparing to single-stranded AS-ODN.
MF technology is a simple, affordable and reproducible method for production of ON-LNPs.
Microfluidics; oligonucleotide; lipid nanoparticles
Liposomes are composed of lipid bilayer membranes that encapsulate an aqueous volume. A major challenge in the development of liposomes for drug delivery is the control of size and size distribution. In conventional methods, lipids are spontaneously assembled into heterogeneous bilayers in a bulk phase. Additional processing by extrusion or sonication is required to obtain liposomes with small size and a narrow size distribution. Microfluidics is an emerging technology for liposome synthesis, because it enables precise control of the lipid hydration process. Here, we describe a number of microfluidic methods that have been reported to produce micro/nanosized liposomes with narrower size distribution in a reproducible manner, focusing on the use of continuous-flow microfluidics. The advantages of liposome formation using the microfluidic approach over traditional bulk-mixing approaches are discussed.
Disulfide-linked oligodeoxyribonucleotide (ODN) liposomes were formulated and evaluated for the delivery of antisense ODN G3139 in KB human oral carcinoma cells.
Materials and Methods
Liposomes composed of 1,2-di-(9Z-octadecenoyl)-3-trimethylammo-nium-propane (DOTAP)/egg phosphatidylcholine/α-tocopheryl polyethylene glycol 1000 succinate were incorporated with hydrophobized disulfide-linked ODN. Disulfide-linked ODN liposomes were characterized for their size, ODN intracellular delivery, Bcl-2 mRNA and protein expression, growth inhibition, and chemosensitization.
Intracellular delivery of ODN with disulfide-linked ODN liposomes was more efficient than that with non-liposomal hydrophobized disulfide-linked ODN. Treatment of the cells with disulfide-linked ODN liposomes resulted in efficient Bcl-2 down-regulation greater than that with hydrophobized disulfide-linked ODN and consistent with that of cellular growth inhibition and the sensitization to daunorubicin in KB cells. Disulfide-linked ODN liposomes exhibited superior colloidal stability during 5-week storage.
Disulfide-linked liposomes are effective delivery vehicles for antisense ODN.
Liposomes; disulfide; antisense oligonucleotide; G3139; Bcl-2; drug delivery
Efficient and site-specific delivery of therapeutic drugs is a critical challenge in clinical treatment of cancer. Nano-sized carriers such as liposomes, micelles, and polymeric nanoparticles have been investigated for improving bioavailability and pharmacokinetic properties of therapeutics via various mechanisms, for example, the enhanced permeability and retention (EPR) effect. Further improvement can potentially be achieved by conjugation of targeting ligands onto nanocarriers to achieve selective delivery to the tumour cell or the tumour vasculature. Indeed, receptor-targeted nanocarrier delivery has been shown to improve therapeutic responses both in vitro and in vivo. A variety of ligands have been investigated including folate, transferrin, antibodies, peptides and aptamers. Multiple functionalities can be incorporated into the design of nanoparticles, e.g., to enable imaging and triggered intracellular drug release. In this review, we mainly focus on recent advances on the development of targeted nanocarriers and will introduce novel concepts such as multi-targeting and multi-functional nanoparticles.
Drug delivery; nanocarriers; drug targeting
Liposomes have been successfully used as delivery vehicles for anticancer drugs. Both sonication and microfluidic technologies have been used to produce liposomes. The combination of the two methods was evaluated in this study.
Materials and Methods
The microfluidic devices, mainly comprising micro-dispensers and a sonicator, were used to produce liposomal nanoparticles. Sonication was used to enhance the reduction of liposome size.
Sonication significantly reduced the size of the liposomes. The particle size also decreased as the buffer to solvent flow rate ratio increased. The smallest particle sizes were achieved with a volumetric flow rate of lipids at 0.374 ml/min.
The microfluidic devices in combination with ultrasound are simple and may be used to produce liposomal nanoparticles with narrow size distribution.
Liposomes; microfluidics; ethanol injection; sonication
While calcium-phosphate has been used to deliver plasmid DNA (pDNA) for decades, the method is typically characterized by low and irreproducible transfection efficiency relative to the other non-viral approaches, such as liposomes and polymers. Here we report a novel gene transfer vector comprising lipid-coated nano-calcium-phosphate (LNCP) that provides consistently efficient and satisfactory pDNA delivery. It is based on core-shell nanoparticles comprising a calcium-phosphate core and a cationic lipid shell. This method, in contrast to the solution precipitation methods used in the past, yields colloidally stable calcium-phosphate nanoparticles inside the cationic liposomes. Our results indicate that the particle size and the size distribution of the LNCP remain virtually unchanged even after 21 days of storage. Atomic force microscopy measurements reveal that the LNCP have a 5-fold higher rigidity than common cationic liposomes. The LNCP transfected pDNA 24 times greater than the naked pDNA and 10-fold greater relative to the standard calcium-phosphate precipitation preparations, suggesting that the LNCP may have potential as a novel transfection agent for gene therapy.
Nanoparticles; Cationic liposome; Calcium-phosphate; Gene therapy
Herein we describe the development and implementation of a nanoporous cell-therapy device with controllable biodegradation. Dopamine-secreting PC12 cells were housed within newly formulated alginate-glutamine degradable polylysine (A-GD-PLL) microcapsules. The A-GD-PLL microcapsules provided a 3-D microenvironment for good spatial cell growth, viability and proliferation. The microcapsules were subsequently placed within a poly(ethylene glycol) (PEG)-coated poly(ε-caprolactone) (PCL) chamber covered with a PEG-grafted PCL nanoporous membrane formed by phase inversion. To enhance PC12 cell growth and to assist in controlled degradation of both the PC12 cells and the device construct, small PCL capsules containing neural growth factor (PCL-NGF) and a poly(lactic-co-glycolic acid) pellet containing glutamine (PLGA-GLN) were also placed within the PCL chamber. Release of NGF from the PCL-NGF capsules facilitated cell proliferation and viability, while the controlled release of GLN from the PLGA-GLN pellet resulted in A-GD-PLL microcapsule degradation and eventual PC12 cell death following a pre-specified period of time (4 weeks in this study). In vivo, our device was found to be well tolerated and we successfully demonstrated the controlled release of dopamine over a period of four weeks. This integrated biodegradable device holds great promise for the future treatment of a variety of diseases.
Nanoporous capsules; Controllable biodegradation; Glutamine-degradable PLL microcapsules; Neural growth factor; Dopamine secretion
Clinical application of small interfering RNA (siRNA) requires safe and efficient delivery in vivo. Here, we report the design and synthesis of lipid nanoparticles (LNPs) for siRNA delivery based on cationic lipids with multiple tertiary amines and hydrophobic linoleyl chains. LNPs incorporating the lipid containing tris(2-aminoethyl)amine (TREN) and 3 linoleyl chain, termed TRENL3, were found to have exceptionally high siRNA transfection efficacy that was markedly superior to lipofectamine, a commercial transfection agent. In addition, inclusion of polyunsaturated fatty acids, such as linoleic acid and linolenic acids in the formulation further enhanced the siRNA delivery efficiency. TRENL3 LNPs were further shown to transported siRNA into the cytosol primarily via macropinocytosis rather than clathrin-mediated endocytosis. The new LNPs have demonstrated preferential uptake by the liver and hepatocellular carcinoma in mice, thereby leading to high siRNA gene silencing activity. These data suggest potential therapeutic applications of TRENL3 mediated delivery of siRNA for liver diseases.
Cationic lipids; Lipid nanoparticles; Small interfering RNA; hepatocellular carcinoma
Exiguobacterium sp. strain 8-11-1 is a newly isolated alkaliphile, which was reported to efficiently produce l-lactate using NaOH as the neutralizing agent. Here, we present the first 2.9-Mb assembly of its genome sequence, which may provide useful information related to its efficient lactate production and sodium ion tolerance capacities.
Reported herein is a medical curiosities vascular tumor primary arising from the kidney and exhibiting unique histopathological features. A 32-year-old woman underwent a total nephrectomy of right kidney because of a mass localized in the inferior pole. Distinct from other vascular lesions, on histology the tumor had a peculiar composite pattern, consisting of benign and malignant vascular components, which were haphazardly intermixed without any definite margins. The malignant component was composed of epithelioid hemangioendothelioma (45%) and angiosarcoma (50%) with moderate differentiation. Immunohistochemically, the oval to cuboidal to spindle tumor cells expressed only endothelial markers (CD31, CD34 and factor VIII-related antigen). And the angiosarcomatous component was characterized by the presence of a greater proliferation index Ki-67. Unlike other epithelial tumors, smooth muscle actin (SMA), cytokeratin, EMA and S-100 were all negative in the epithelioid tumor cells. These findings led to the diagnosis of a low-grade vascular neoplasm with morphological features consistent with so-called composite hemangioendothelioma (CHE). At 11 month follow up the patient was alive, without evidence of tumor recurrence. CHE is an extremely rare vascular neoplasm, with borderline malignant potential, which mostly occurs in distal extremity of the limbs at the cutaneous level and, only 30 cases have been previously described until now. To our knowledge, this is the first report of CHE arising from the kidney and widens the spectrum of primary vascular tumors arising in the kidney.
Composite hemangioendothelioma; hemangioendothelioma; rare tumors; kidne
In this study, we presented an additional case of renal hemangioblastoma, which demonstrates PAX2 and focal CD10 expression. Histologically, the tumor consisted of sheets of oval or polygonal cells and a prominent vascular network. The tumor cells varied in size, and possessed pale or eosinophilic cytoplasm that sometimes contained sharply delineated fine vacuoles. The tumor cell nuclei with inconspicuous nucleoli showed moderate nuclear atypia and pleomorphism. Focal areas of stromal hyalinization and sclerosis were detected. On account of its strong or moderate immunoreactivity for the a-inhibin, S100, NSE, and EGFR, the diagnosis of renal hemangioblastoma was established. For further evidence of VHL deficiency, the tumor was subjected to VHL sequence analysis of all three exons and fluorescence in situ hybridization (FISH) detection for chromosome 3p deletion. None of the VHL gene mutations and chromosome 3p deletion was detected in the tumor. Because of several shared morphological and immunophenotypic features, renal hemangioblastoma may be underrecognized and should be included in the differential diagnosis of primary renal tumors, in particular clear cell renal cell carcinoma. The unexpected positive staining of PAX2 and CD10 in renal hemangioblastoma should be particular concerned. Using a combination of immunoprofile may be helpful to the differential diagnosis of these renal tumors.
Kidney; hemangioblastoma; PAX2; CD10; von-Hippel-Lindau (VHL); fluorescence in situ ridization (FISH); molecular genetics; differential diagnosis
Escherichia coli NusA, an essential component of the RNA polymerase elongation complex, is involved in transcriptional elongation, termination, anti-termination, cold shock and stress-induced mutagenesis. In this study, we demonstrated that NusA can self-assemble into oligomers under heat shock conditions and that this property is largely determined by the C-terminal domain. In parallel with the self-assembly process, NusA also acquires chaperone activity. Furthermore, NusA overexpression results in the enhanced heat shock resistance of host cells, which may be due to the chaperone activity of NusA. Our results suggest that E. coli NusA can act as a protector to prevent protein aggregation under heat stress conditions in vitro and in the NusA-overexpressing strain. We propose a new hypothesis that NusA could serve as a molecular chaperone in addition to its functions as a transcription factor. However, it remains to be further investigated whether NusA has the same function under normal physiological conditions.
Shigella flexneri is the major cause of bacterial shigellosis in developing countries. S. flexneri is divided into at least 19 serotypes, the majority of which are modifications of the same basic O-antigen by glucosylation and/or O-acetylation of its sugar residues by phage encoded serotype-converting genes. Recently, a plasmid encoded phosphoethanolamine (PEtN) modification of the O-antigen has been reported, which is responsible for the presence of the MASF IV-1 determinant and results in conversion of traditional serotypes X, 4a and Y to novel serotypes Xv, 4av and Yv, respectively. In this study, we characterized 19 serotype Yv strains isolated in China. A variant of the O-antigen phosphoethanolamine transferase gene opt (formerly called lpt-O) carried by a pSFxv_2-like plasmid was found in serotype Yv strains, which specifies the phosphorylation pattern on the O-antigen of this serotype. For the majority of the O-antigen units, the PEtN modification occurs on RhaIII, while for a minority, modifications occur on both RhaII and RhaIII. Serotype-specific gene detection and PFGE analysis suggested that these serotype Yv isolates were originated from serotypes Y, Xv and 2a by acquisition of an opt-carrying plasmid and/or inactivation of serotype-specific gene gtrII or gtrX. These data, combined with those of serotypes Xv and 4av reported earlier, demonstrate that the plasmid-encoded PEtN modification is an important serotype conversion mechanism in S. flexneri, in addition to glucosylation and O-acetylation.
It has been supposed that green tea polyphenols (GTPs) have neuroprotective effects on brain damage after brain ischemia in animal experiments. Little is known regarding GTPs’ protective effects against the blood-brain barrier (BBB) disruption after ischemic stroke. We investigated the effects of GTPs on the expression of claudin-5, occludin, and ZO-1, and the corresponding cellular mechanisms involved in the early stage of cerebral ischemia.
Male Wistar rats were subjected to a middle cerebral artery occlusion (MCAO) for 0, 30, 60, and 120 min. GTPs (400 mg/kg/day) or vehicle was administered by intragastric gavage twice a day for 30 days prior to MCAO. At different time points, the expression of claudin-5, occludin, ZO-1, and PKCα signaling pathway in microvessel fragments of cerebral ischemic tissue were evaluated.
GTPs reduced BBB permeability at 60 min and 120 min after ischemia as compared with the vehicle group. Transmission electron microscopy also revealed that GTPs could reverse the opening of tight junction (TJ) barrier at 60 min and 120 min after MACO. The decreased mRNA and protein expression levels of claudin-5, occludin, and ZO-1 in microvessel fragments of cerebral ischemic tissue were significantly prevented by treatment with GTPs at the same time points after ischemia in rats. Furthermore, GTPs could attenuate the increase in the expression levels of PKCα mRNA and protein caused by cerebral ischemia.
These results demonstrate that GTPs may act as a potential neuroprotective agent against BBB damage at the early stage of focal cerebral ischemia through the regulation of TJ and PKCα signaling.
Green tea polyphenols; Cerebral ischemia; Blood–brain barrier; Tight junction; Protein kinase Cα
AIM: To explore the role of CDX2 in the multi-drug resistance (MDR) process of gastric cancer in vitro and in vivo.
METHODS: A cisplatin-resistant gastric cancer cell line with stable downregulation of CDX2 was established. mRNA and protein expression levels of CDX2, survivin, cyclin D1, and c-Myc were detected by western blotting and semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The influence of downregulation of CDX2 on MDR was assessed by measuring IC50 of SGC7901/DDP cells to cisplatin, doxorubicin, and 5-fluorouracil, rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. In addition, we determined the in vivo effects of CDX2 small interfering RNA (siRNA) on tumor size, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining.
RESULTS: CDX2 siRNA led to downregulation of endogenous CDX2 mRNA (0.31 ± 0.05 vs 1.10 ± 0.51, 0.31 ± 0.05 vs 1.05 ± 0.21, P = 0.003) and protein (0.12 ± 0.08 vs 0.51 ± 0.07, 0.12 ± 0.08 vs 0.55 ± 0.16, P = 2.57 × 10-4) expression. It significantly promoted the sensitivity of SGC7901/DDP cells to cisplatin (0.12 ± 0.05 vs 0.33 ± 0.08, 0.12 ± 0.05 vs 0.39 ± 0.15, P = 0.001), doxorubicin (0.52 ± 0.13 vs 4.11 ± 1.25, 0.52 ± 0.13 vs 4.05 ± 1.44, P = 2.81 × 10-4), and 5-fluorouracil (0.82 ± 0.13 vs 2.81 ± 0.51, 0.82 ± 0.13 vs 3.28 ± 1.03, P = 1.71 × 10-4). Flow cytometry confirmed that the percentage of apoptotic cells increased after CDX2 downregulation (32.15% ± 2.15% vs 17.63% ± 3.16%, 32.15% ± 2.15% vs 19.3% ± 2.25%, P = 1.73 × 10-6). This notion was further supported by the observation that downregulation of CDX2 blocked entry into the S-phase of the cell cycle (31.53% ± 3.78% vs 65.05% ± 7.25%, 31.53% ± 3.78% vs 62.27% ± 5.02%, P = 7.55 × 10-7). Furthermore, downregulation of CDX2 significantly increased intracellular accumulation of doxorubicin (0.21 ± 0.06 vs 0.41 ± 0.11, 0.21 ± 0.06 vs 0.40 ± 0.08, P = 0.003). In molecular studies, semiquantitative RT-PCR and western blotting revealed that CDX2 downregulation could inhibit expression of c-Myc, survivin and cyclin D1.
CONCLUSION: CDX2 may be involved in regulating multiple signaling pathways in reversing MDR, suggesting that CDX2 may represent a novel target for gastric cancer therapy.
Homeobox gene CDX2; RNA interference; Gastric cancer; Drug resistance; Murine model
Human bone marrow mesenchymal stem/stromal cells (MSCs) are multipotent progenitor cells with multilineage differentiation potentials including osteogenesis and adipogenesis. While significant progress has been made in understanding transcriptional controls of MSC fate, little is known about how MSC differentiation is epigenetically regulated. Here we show that the histone demethylases KDM4B and KDM6B play critical roles in osteogenic commitment of MSCs by removing H3K9me3 and H3K27me3. Depletion of KDM4B or KDM6B significantly reduced osteogenic differentiation and increased adipogenic differentiation. Mechanistically, while KDM6B controlled HOX expression by removing H3K27me3, KDM4B promoted DLX expression by removing H3K9me3. Importantly, H3K27me3- and H3K9me3-positive MSCs of bone marrow were significantly elevated in ovariectomized and aging mice in which adipogenesis was highly active. Since histone demethylases are chemically modifiable, KDM4B and KDM6B may present as novel therapeutic targets for controlling MSC fate choices, and lead to clues for new treatment in metabolic bone diseases such as osteoporosis.
DOTAP, as a racemic mixture, is a cationic lipid and a widely used transfection reagent. In this study, the effect of DOTAP’s stereochemical structure on transfection efficiency was evaluated in vitro. Racemic and enantiomerically pure DOTAP were used in lipoplex formulations to deliver siRNA to MCF-7 cells, targeting the aromatase enzyme. At the 50nM siRNA concentration and lipid-to-RNA charge ratios of 4 and 5, the R enantiomer of DOTAP was found to perform better than either the S- and the racemic agent. In addition, at 10 nM siRNA concentration and a charge ratio of 3, the R- lipoplex formulation silenced aromatase by ~ 50% whereas the S and racemic formulations caused no significant target downregulation. Differences in lipid packing were modeled using membrane simulations. The results showed that, when combined with cholesterol, pure R-DOTAP and S-DOTAP enantiomers had 105% and 115% of lipid density relative to racemic DOTAP, respectively. These findings suggest an important role of lipid chirality in future development of lipid based siRNA delivery systems.
DOTAP; enantiomer; siRNA; lipoplex; aromatase; modeling
Resolvin D1 (RvD1) is a lipid-derived mediator generated during the resolution inflammation. While the immunoresolvent effects of Resolvins have been extensively studied in leukocytes, actions of Resolvins on intrinsic kidney cells have received little attention. The podocyte plays a central role in glomerular function, and podocyte damage can lead to proteinuria and glomerulosclerosis. This study examined whether RvD1 has renoprotective effects upon podocytes. We investigated a mouse model of adriamycin (ADR) nephropathy featuring rapid induction of podocyte damage and proteinuria followed by glomerulosclerosis. We identified a progressive loss of synaptopodin expression over a 28 day time-course of ADR nephropathy which was associated with increased acetylation of 14-3-3β and reduced synaptopodin phosphorylation. Groups of mice were given once daily RvD1 treatment (4 ng/g body weight/day) starting either 30 min (early treatment) or 14 days (late treatment) after ADR injection and continued until mice were killed on day 28. Early, but not late, RvD1 treatment attenuated ADR-induced proteinuria, glomerulosclerosis and tubulointerstitial fibrosis, modified macrophages from an M1 to M2 phenotype. Early RvD1 treatment prevented the down-regulation of synaptopodin expression and changes in 14-3-3β acetylation and synaptopodin phosphorylation. In a podocyte cell line, RvD1 was shown to prevent rapid TNF-α-induced down-regulation of synaptopodin expression. In transfection studies, TNF-α-induced a decrease in synaptopodin phosphorylation and an increase in acetylation of 14-3-3β, resulting in disassociation between 14-3-3β and synaptopodin. RvD1 prevented TNF-α induced post-translational modification of synaptopodin and 14-3-3β proteins, and maintained the synaptopodin/14-3-3β interaction. Furthermore, replacement of lysine K51, or K117+K122 in 14-3-3β with glutamine, to mimic lysine acetylation, significantly reduced the interaction between 14-3-3β and synaptopodin. In conclusion, our studies provide the first evidence that RvD1 can protect against podocyte damage by preventing down-regulation of synaptopodin through inhibition of 14-3-3β/synaptopodin dissociation. RvD1 treatment may have potential application in the treatment of chronic kidney disease.
Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4+CD25+ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4+CD25+Foxp3+ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4+CD25+Foxp3+ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.
Papillary renal cell carcinoma (PRCC) is traditionally classified into type 1 and type 2. Recently, an oncocytic variant of PRCC has been described. We report a series of 6 oncocytic renal papillary tumors (OPRCC) which tended to occur in older patients (mean, 56.8 years) with a male preference (male-to-female ratio is 5:1). All 6 patients are alive with no evidence of disease after initial resection, showing an indolent clinical behavior. Histologically, tumors exhibited predominant papillary structure with delicate fibrovascular cores. Papillae were lined by single layers of cells with large, deeply eosinophilic and finely granular cytoplasms and round regular nucleus. The phagocytosis of tumor cells was frequently and evidently seen in our cases that hemosiderin-laden tumor cells and foamy tumor cells were noticed in five and four cases respectively. All tumors were immunoreactive for racemase, vimentin, CD10, and MET and negative for CD117. While E-cadherin, EMA, and cytokeratin 7 exhibited variable immunopositivity. FISH analysis was performed in five of six cases and found heterogeneous results. Trisomy of chromosomes 7 was found in three cases and trisomy of chromosomes 17 in two cases. Loss of chromosome Y was noted in one of four tumors in male patients. MET gene status was also investigated by direct sequencing in all 6 cases and found no distinct mutation in any case. These results suggest that OPRCC shows distinct morphology, indolent clinical behavior, and similar immunohistochemical and cytogenetic features with PRCC, seems to be a variant in the PRCC group. Whether the strong expression of MET indicates a potential therapeutic target is still unknown and requires further investigation in clinical trials.
Kidney; papillary renal cell carcinoma; oncocytic tumors; MET
Shiga toxin (Stx)-producing Escherichia coli (STEC) are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx1 and stx2 genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96%) stx1 and/or stx2-positive samples, 85 (61.59%) were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE) and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx1 subtype (stx1a) and 5 stx2 subtypes (stx2a, stx2b, stx2c, stx2d and stx2g) were present in these STEC isolates. Apart from lpfAO157/OI-141, lpfAO157/OI-154, lpfAO113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP) were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.
The retina is a light-sensitive tissue of the central nervous system that is vulnerable to ischemia. The pathological mechanism underlying retinal ischemic injury is not fully understood. The purpose of this study was to investigate structural and functional changes of different types of rat retinal neurons and visual behavior following transient global ischemia. Retinal ischemia was induced using a 4-vessel occlusion model. Compared with the normal group, the number of βIII-tubulin positive retinal ganglion cells and calretinin positive amacrine cells were reduced from 6 h to 48 h following ischemia. The number of recoverin positive cone bipolar cells transiently decreased at 6 h and 12 h after ischemia. However, the fluorescence intensity of rhodopsin positive rod cells and fluorescent peanut agglutinin positive cone cells did not change after reperfusion. An electroretinogram recording showed that the a-wave, b-wave, oscillatory potentials and the photopic negative response were completely lost during ischemia. The amplitudes of the a- and b-waves were partially recovered at 1 h after ischemia, and returned to the control level at 48 h after reperfusion. However, the amplitudes of oscillatory potentials and the photopic negative response were still reduced at 48 h following reperfusion. Visual behavior detection showed there was no significant change in the time spent in the dark chamber between the control and 48 h group, but the distance moved, mean velocity in the black and white chambers and intercompartmental crosses were reduced at 48 h after ischemia. These results indicate that transient global ischemia induces dysfunction of retinal ganglion cells and amacrine cells at molecular and ERG levels. However, transient global ischemia in a 17 minute duration does not appear to affect photoreceptors.
Metastatic melanoma, the primary cause of skin cancer-related death, warrants new therapeutic approaches that target the regulatory machinery at molecular level. While long noncoding RNAs (lncRNAs) are dysregulated in a number of cancer types, limited data are available on the expression and function of lncRNAs in melanoma metastasis. The primary objective of this study was to investigate the role of 6 metastasis-related lncRNAs in pairs of primary melanoma and matched lymph node metastatic tissues. Among the tested lncRNAs, HOTAIR was the most highly expressed in lymph node metastasis. The role of HOTAIR in melanoma cell motility and invasion was further evaluated by knocking down HOTAIR with siRNAs. Knockdown of HOTAIR resulted in the reduction of motility and invasion of human melanoma cell line A375, as assessed by wound healing assay and Matrigel-based invasion assay. siHOTAIR also suppressed the degradation of gelatin matrix, suggesting that HOTAIR promotes gelatinase activity. Together, our study shows that HOTAIR is overexpressed in metastatic tissue, which is associated with the ability of HOTAIR to promote melanoma cell motility and invasion. These data indicate that lncRNAs may be involved in the metastasis of melanoma and provide support for further evaluation of lncRNAs in melanoma.
Melioidosis, caused by Burkholderia pseudomallei, is considered to be endemic to Northern Australia and Southeast Asia, with high mortality and relapse rates, regardless of powerful antibiotic therapy. Here we report the first genome sequence of Burkholderia pseudomallei strain BPC006, obtained from a melioidosis patient in Hainan, China. The genome sizes of the 2 chromosomes were determined to be 4,001,777 bp and 3,153,284 bp.