[Effects of ApoE gene polymorphism on the efficacy of atorvastatin in the treatment of hyperlipidemia].

Objective: To study the role of ApoE gene polymorphism on efficacy of atorvastatin in lowering the lipid and its clinical significance. Methods: A total of 962 patients with hypercholesterolemia were selected between January 1 st and December 31 st 2014. The ApoE genepolymorphism in patients with hyperlipidemia was performed by using polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) method in translational medicine center of Huaihe Hospital. Patients with ApoE genotype E3/3 and E3/4 were selected and treated with atorvastatin 10 mg/d for 4 weeks. Before and after treatment, triglycerides (TG) and total cholesterol (TC) was detected by enzyme colorimetry method. High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were performed by Clearance method. Lipoprotein(a) (Lp(a)) was performed by turbidimetric inhibition immunoassay. ApoE gene expression was performed by real-time PCR. Results: In the 6 gene types, the frequencies of E3/4 and E3/3 were 30.6% (294 cases) and 59.1% (569 cases) respectively. After treatment with atorvastatin, the change percent of TC, LDL-C, HDL-C, TG, Lp(a) in E3/4 and E3/3 group were -(23.0±4.7)% vs -(12.0±3.1)% (P<0.001), -(33.0±4.8)% vs -(20.0±3.9)% (P<0.001), (18.0±3.8)% vs (6.0±2.6)% (P<0.001), -(23.0±3.9)% vs -(13.0±2.7)% (P<0.001), -(21.5±4.5)% vs -(20.9±4.0)% (P=0.054), respectively. ApoE gene expression in E3/3 and E3/4 groups were down-regulated in both groups, and the change in E3/3 group was obvious than that of E3/4 group. Conclusion: After treatment with atorvastatin, levels of lipids and ApoE gene expression in ApoE genotype E3/3 patients decreased, which were more evident than E3/4 patients.

IL-16 rs4778889 polymorphism contribution to the development of renal cell cancer in a Chinese population.

IL-16 plays an important role in affect the secretion of tumor-related inflammatory cytokines. We aimed to assess the role of interleukin-16 (IL-16) rs4778889 T/C and rs11556218 T/G polymorphisms in the occurrence of renal cell cancer (RCC). This study is composed of 274 RCC patients and 274 control subjects. Genotyping of polymorphisms was performed using polymerase chain reaction combined with restriction fragment length polymorphism analysis. All statistical analysis was carried out by the SPSS statistical software package, version 16.0 (SPSS Inc., Chicago, IL, USA). Using conditional logistic regression analysis, the TC and CC genotypes of rs4778889 exhibited a higher risk of RCC, with adjusted ORs (and 95%CIs) of 1.79 (1.23-2.62) and 2.67 (1.29-5.69), respectively. Moreover, under dominant and recessive models, individuals carried the rs4778889 polymorphism was exhibited elevated RCC risk, with adjusted ORs (and 95%CI) of 1.93 (1.35-2.76) and 2.11 (1.05-4.45), respectively. No significant differences were observed in rs11556218 genotype frequencies between the study groups. In conclusion, the results of our study reveal an association between the IL-16 rs4778889 polymorphism and heightened risk of RCC.

Effects of Tripterygium wilfordii glycosides on regulatory T cells and Th17 in an IgA nephropathy rat model.

In this study, we examined the effects of Tripterygium wilfordii glycosides (TWGs) on Th17 and regulatory T cells (Tregs) in an immunoglobulin A nephropathy (IgAN) rat model. IgAN model rats were randomly divided into the model group, TWG treatment group, and prednisone group. Normal rats were included as controls. There were 6 rats in each group. The urine protein levels and the number of red blood cells in urine were analyzed at 24 h. IgA deposition in renal tissue was detected by fluorescence microscopy. The concentration of interleukin-17 in serum was detected by an enzyme-linked immunosorbent assay and the number of Tregs in blood was analyzed by flow cytometry. TWGs and prednisone significantly reduced urine protein levels and urine red blood cells at 24 h in IgAN model rats (P < 0.01), but prednisone had a greater effect than did TWGs (P < 0.05). TWGs and prednisone reduced IgA deposition in renal tissue, but prednisone had a greater effect than TWGs. T. wilfordii glycosides and prednisone significantly decreased the serum IL-17 level in an IgAN rat model and increased the number of Tregs in the blood (P < 0.01). There was no significant difference between prednisone and TWGs (P > 0.05). In conclusion, TWGs had therapeutic effects on IgAN model rats and may regulate the immune balance of Th17 and Tregs.

Trends in the inactive kidney transplant waitlist and implications for candidate survival.

In November 2003, OPTN policy was amended to allow kidney transplant candidates to accrue waiting time while registered as status 7, or inactive. We evaluated trends in inactive listings and the association of inactive status with transplantation and survival, studying 262,824 adult first-time KT candidates listed between 2000 and 2011. The proportion of waitlist candidates initially listed as inactive increased from 2.3% prepolicy change to 31.4% in 2011. Candidates initially listed as inactive were older, more often female, African American, and with higher body mass index. Postpolicy change, conversion from initially inactive to active status generally occurred early if at all: at 1 year after listing, 52.7% of initially inactive candidates had been activated; at 3 years, only 66.3% had been activated. Inactive status was associated with a substantially higher waitlist mortality (aHR 2.21, 95%CI:2.15-2.28, p<0.001) and lower rates of eventual transplantation (aRR 0.68, 95%CI:0.67-0.70, p<0.001). In summary, waitlist practice has changed significantly since November 2003, with a sharp increase in the number of inactive candidates. Using the full waitlist to estimate organ shortage or as a comparison group in transplant outcome studies is less appropriate in the current era.

MSC-seeded dense collagen scaffolds with a bolus dose of VEGF promote healing of large bone defects.

The functional repair of large skeletal defects remains a significant challenge to orthopaedic surgeons due to the lack of effective strategies to promote bone regeneration, particularly in the elderly. This study investigated the potential use of bone marrow derived mesenchymal stromal cells (MSC) in a dense collagen scaffold with a bolus dose of vascular endothelial growth factor (VEGF) to repair a defect in the femoral diaphysis of mice. MSC isolated from bone marrow of 4-month-old donor mice were seeded in type I collagen gels that were then compressed to form scaffolds with a fibrillar density similar to osteoid. The cells remained metabolically active in scaffolds incubated in vitro for up to 15 days and differentiated into osteoblasts that deposited calcium-phosphate mineral into the scaffold, which was quantified using micro-computed tomographic (micro-CT) imaging. When implanted in a 1 mm x 3 mm unicortical defect the MSC-loaded scaffolds were rapidly mineralised and integrated into host bone with administration of 10 ng of recombinant VEGF injected into the femoral canal at 4 days postoperative. Empty scaffolds and MSC-seeded scaffolds implanted in defects that did not receive a bolus dose of VEGF did not mineralise or integrate with native bone. The approach with MSC, hydrogels and a biologic factor already approved for human use warrants further pre-clinical investigation with a large animal model.

Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule.

Human leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the alpha 1 domain of various HLA-B and -C molecules. A CD4+ CD8- cytotoxic T cell line, CTL-AV, that is specific for the HLA-B7 peptide presented by HLA-DR11.1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11.1, did not present the HLA-B7 peptide to CTL-AV. Peptides from the alpha 1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11.1 was killed by CTL-AV in the absence of any added HLA-B7 peptide. The processing and presentation of HLA-B7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not. Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11.1+, HLA-B7- cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTL-AV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway of antigen presentation. They also emphasize the capacity for presentation of MHC peptides by MHC molecules.

Apparent lack of MHC restriction in binding of class I HLA molecules to solid-phase peptides.

The specificity of binding of solubilized, purified HLA-A,B molecules to solid-phase peptides has been examined using the assay described by Bouillet et al. [1989. Nature (Lond.). 339:473.] 64 peptides derived from the sequences of viral antigens, HLA-A,B,C heavy chains, and clathrin light chains were tested for binding to HLA-A2.1, Aw68.1, Aw69, B44, and B5, molecules that differ by 5-17 residues of the peptide binding groove. 15 of the peptides, including those known to be T cell epitopes, gave significant binding. The pattern of peptide binding for each of the five HLA-A,B molecules was not significantly different. Binding was demonstrated to be a property of native beta 2m-associated HLA-A,B molecules that preserved conformational antigenic determinants after binding to peptide. In comparison to our previous results from solution-based assays the proportion of HLA-A,B molecules that can bind solid-phase peptides is very high. This accessibility of solid-phase peptides to the binding site of MHC molecules may be directly related to the observed absence of MHC specificity in the binding.

LncRNA MALAT1 inhibits osteogenic differentiation of mesenchymal stem cells in osteoporosis rats through MAPK signaling pathway.

OBJECTIVE: The aim of this study was to explore whether long non-coding ribonucleic acid metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) could lead to osteoporosis (OP) by stimulating the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. MATERIALS AND METHODS: The OP model was first successfully established in rats. The expression of lncRNA MALAT1 in OP rats and normal rats was detected via quantitative Polymerase Chain Reaction (qPCR). Bone marrow mesenchymal stem cells (BMSCs) were cultured and transfected to establish the MALAT1 knockdown model. Subsequently, the apoptosis of mesenchymal stem cells in MALAT1 siRNA group and NC siRNA group was detected via flow cytometry. Meanwhile, the expressions of the MAPK signaling pathway proteins related to OP were detected via Western blotting. After alkaline phosphatase (ALP) staining in cells of both groups, early osteogenic differentiation of BMSCs was observed. RESULTS: The results of qPCR showed that the expression of lncRNA MALAT1 in OP rats was significantly lower than that of normal rats. It was observed under a fluorescence microscope that there were a large number of siRNA particles in BMSCs. The expression of lncRNA MALAT1 in cells was detected via Real Time-fluorescence qPCR as well. The results indicated that siRNA transfection could effectively inhibit the expression of lncRNA MALAT1, indicating successful transfection. Flow cytometry revealed that no significant difference was observed in the apoptosis of BMSCs between the MALAT1 siRNA group and NC siRNA group. Besides, the results of Western blotting showed that the expression levels of the MAPK signaling pathway-related proteins extracellular signal-regulated kinase 1/2 (ERK1/2) and P38 in MALAT1 siRNA group were significantly higher than those of the NC siRNA group. This indicated that inhibiting the expression of lncRNA MALAT1 might promote the activation of the OP-related MAPK pathway. According to the results of ALP staining, the depth of staining in MALAT1 siRNA group was markedly declined when compared with the NC siRNA group. Quantification of ALP activity demonstrated that ALP activity in the MALAT1 siRNA group was markedly declined compared with the NC siRNA group. The above results suggested that suppressing the expression of lncRNA MALAT1 could reduce the ALP activity of BMSCs. Furthermore, lncRNA MALAT1 inhibited osteogenic differentiation of BMSCs. CONCLUSIONS: LncRNA MALAT1 was lowly expressed in OP rats. Moreover, it inhibited osteogenic differentiation of BMSCs by enhancing the activation of the MAPK signaling pathway, thereby promoting OP progression.

Selective dorsal rhizotomy in ambulant children with cerebral palsy.

BACKGROUND: Selective dorsal rhizotomy (SDR) is a surgical procedure for treating spasticity in ambulant children with cerebral palsy (CP). However, controversies remain regarding indications, techniques and outcomes. CURRENT EVIDENCE SUMMARY: Because SDR is an irreversible procedure, careful patient selection, a multi-disciplinary approach in assessment and management and division of the appropriate proportion of dorsal rootlets are felt to be paramount for maximizing safety. Reliable evidence exists that SDR consistently reduces spasticity, in a predictable manner and to a substantial degree. However, functional improvements are small in the short-term with long-term benefits difficult to assess. FUTURE OUTLOOK: There is a need for high-quality studies utilizing long-term functional outcomes and well-matched control groups. Collaborative, multicentre efforts are required to further define the role of SDR as part of the management paradigm in maximizing physical function in spastic CP.

Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress.

Shear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress-induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress-induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate-responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH2-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress-induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

Analysis of ATF3, a transcription factor induced by physiological stresses and modulated by gadd153/Chop10.

We demonstrate that ATF3, a member of the ATF/CREB family of transcription factors, is induced in a variety of stressed tissues: mechanically injured liver, toxin-injured liver, blood-deprived heart, and postseizure brain. We also demonstrate that an ATF3-interacting protein, gadd153/Chop10, forms a nonfunctional heterodimer with ATF3: the heterodimer, in contrast to the ATF3 homodimer, does not bind to the ATF/cyclic AMP response element consensus site and does not repress transcription. Interestingly, ATF3 and gadd153/Chop10 are expressed in inverse but overlapping manners during the liver's response to carbon tetrachloride (CCl4): the level of gadd153/Chop10 mRNA is high in the normal liver and greatly decreases upon CCl4 treatment; the level of ATF3 mRNA, on the other hand, is low in the normal liver and greatly increases upon CCl4 treatment. We hypothesize that in nonstressed liver, gadd153/Chop10 inhibits the limited amount of ATF3 by forming an inactive heterodimer with it, whereas in CCl4-injured liver, the synthesis of gadd153/Chop10 is repressed, allowing the induced ATF3 to function.

gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3.

Recently, we demonstrated that the function of ATF3, a stress-inducible transcriptional repressor, is negatively regulated by a bZip protein, gadd153/Chop10. In this report, we present evidence that ATF3 can repress the expression of its own inhibitor, gadd153/Chop10. First, ATF3 represses a chloramphenicol acetyltransferase reporter gene driven by the gadd153/Chop10 promoter when assayed by a transfection assay in vivo and a transcription assay in vitro. Second, the gadd153/Chop10 promoter contains two functionally important binding sites for ATF3: an AP-1 site and a C/EBP-ATF composite site, a previously unidentified binding site for ATF3. The absence of either site reduces the ability of ATF3 to repress the promoter. Third, overexpression of ATF3 by transient transfection results in a reduction of the endogenous gadd153/Chop10 mRNA level. Fourth, as described previously, ATF3 is induced in the liver upon CCl4 treatment. Intriguingly, we show in this report that gadd153/Chop10 mRNA is not present in areas where ATF3 is induced. Taken together, these results strongly suggest that ATF3 represses the expression of gadd153/Chop10. The mutual negative regulation between ATF3 and gadd153/Chop10 is discussed.

Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines.

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

Spontaneous tumor development in bone marrow-rescued DNA-PKcs(3A/3A) mice due to dysfunction of telomere leading strand deprotection.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at the Thr2609 cluster is essential for its complete function in DNA repair and tissue stem cell homeostasis. This phenomenon is demonstrated by congenital bone marrow failure occurring in DNA-PKcs(3A/3A) mutant mice, which require bone marrow transplantation (BMT) to prevent early mortality. Surprisingly, an increased incidence of spontaneous tumors, especially skin cancer, was observed in adult BMT-rescued DNA-PKcs(3A/3A) mice. Upon further investigation, we found that spontaneous γH2AX foci occurred in DNA-PKcs(3A/3A) skin biopsies and primary keratinocytes and that these foci overlapped with telomeres during mitosis, indicating impairment of telomere replication and maturation. Consistently, we observed significantly elevated frequencies of telomere fusion events in DNA-PKcs(3A/3A) cells as compared with wild-type and DNA-PKcs-knockout cells. In addition, a previously identified DNA-PKcs Thr2609Pro mutation, found in breast cancer, also induces a similar impairment of telomere leading-end maturation. Taken together, our current analyses indicate that the functional DNA-PKcs T2609 cluster is required to facilitate telomere leading strand maturation and prevention of genomic instability and cancer development.

Effects of amiodarone and its active metabolite desethylamiodarone on the ventricular defibrillation threshold.

OBJECTIVES: We evaluated whether the reported difference in the ventricular defibrillation threshold (DFT) between short-term intravenous and oral amiodarone is due to the effect of amiodarone's active metabolite desethylamiodarone (DEA). BACKGROUND: Amiodarone is frequently used in patients with implantable cardioverter-defibrillator devices (ICD). Long-term oral amiodarone raises the DFT, but intravenous amiodarone has not been shown to have this effect. DEA, an active metabolite of amiodarone, has different electrophysiologic properties than its parent compound and may be responsible for the observed different effects of intravenous and oral amiodarone on DFT. METHODS: We ascertained the DFT in 24 pigs randomized to receive intravenous amiodarone, DEA or vehicle. Defibrillation was delivered through a transvenous lead system using a biphasic waveform. The DFT was determined using an up-down DFT algorithm and defined as the average minimal energies resulting in successful defibrillation delivered from ascending and descending serial shocks. RESULTS: Amiodarone caused a dose-response increase in DFT (mean +/- SD) from 22.7 +/- 4.1 (baseline) to 26.1 +/- 2.9 (10 mg/kg body weight), p = 0.11, to 34.9 +/- 8.2 J (after an additional 15 mg/kg), p = 0.035. DEA (10 mg/kg) caused an increase in DFT from 20.5 +/- 6.3 to 33.9 +/- 13.6 J, p < 0.01. Addition of 15 mg/kg of DEA resulted in hemodynamic instability and thus DFT was not obtained. In the control group, DFT decreased from 26.8 +/- 7.7 at baseline to 23.1 +/- 7.4 (dose 1), p = 0.19, to 22.8 +/- 6.2 J (dose 2), p = 0.18. CONCLUSIONS: DEA increases DFT by a greater amount than its parent drug amiodarone. There is an effect of intravenous amiodarone on DFT that is dose dependent.

Cytokine-mobilized peripheral blood CD34+Thy-1+Lin- human hematopoietic stem cells as target cells for transplantation-based gene therapy.

Gene-therapy of blood-borne disorders may be best achieved using hematopoietic stem cells (HSC) which have extensive self renewal potential as well as multilineage repopulating potential as a cellular target. The human HSC, which is CD34+Thy-1+Lin- has been isolated from fetal, adult bone marrow and cytokine-mobilized peripheral blood (MPB) (1-3). Results presented in this study show that the degree of mobilization of HSC into peripheral blood of cancer patients is highly variable and that the combined use of high dose chemotherapy and GM-CSF as a mobilization strategy is superior to the use of G-CSF with regard to the mobilization of true HSC. A multistep cell isolation procedure has been developed which utilizes high speed flow-cytometric cell sorting and allows the isolation of sufficient numbers of HSC from MPB to permit their use as an hematopoietic graft for clinical transplantation. Hematopoietic stem cells isolated from MPB are capable of self-renewal and differentiation into multiple hematopoietic lineages as shown by their behavior in both in vitro and in vivo assays. Mobilized PB mononuclear cells isolated from cancer patients are frequently contaminated with tumor cells. Using this cell isolation procedure, HSC preparations from patients with multiple myeloma have been created with greatly reduced tumor cell burdens. These CD34+Thy-1+Lin- cells are capable of being stably transduced at high efficiency (32-75%) by co-culture on a cell line producing recombinant retroviruses containing the neomycin-resistant gene. These HSC cell populations are likely ideal targets for hematopoietic cell-based gene therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line.

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.

DNA microarray analysis of gene expression in endothelial cells in response to 24-h shear stress.

The recently developed DNA microarray technology provides a powerful and efficient tool to rapidly compare the differential expression of a large number of genes. Using the DNA microarray approach, we investigated gene expression profiles in cultured human aortic endothelial cells (HAECs) in response to 24 h of laminar shear stress at 12 dyn/cm(2). This relatively long-term shearing of cultured HAECs led to the modulation of the expression of a number of genes. Several genes related to inflammation and EC proliferation were downregulated, suggesting that 24-h shearing may keep ECs in a relatively noninflammatory and nonproliferative state compared with static cells. Some genes were significantly upregulated by the 24-h shear stress; these includes genes involved in EC survival and angiogenesis (Tie2 and Flk-1) and vascular remodeling (matrix metalloproteinase 1). These results provide information on the profile of gene expression in shear-adapted ECs, which is the case for the native ECs in the straight part of the aorta in vivo.

Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host.

We have constructed two convenient vectors to produce foreign proteins in Escherichia coli. The first vector was developed to produce histidine (His)-tagged fusion proteins. In addition to encoding six contiguous His residues, it contains three unique restriction sites that allow cloning of a blunt-ended DNA fragment in three different reading frames. Therefore, one can clone any gene of interest in this vector to make a fusion protein tagged with six His at the N terminus. The His-tag allows purification of the fusion protein to almost homogeneity by a nickel-chelating column in a single step. The second vector is a derivative of the first vector; it encodes two tandem phosphorylation sites for heart muscle kinase (HMK) immediately downstream from the His residues. Therefore, the resulting fusion protein can be radiolabeled using [gamma-32P]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.

Presentation of soluble antigen to human T cells by products of multiple HLA-linked loci: analysis of antigen presentation by a panel of cloned, autologous, HLA-mutant Epstein-Barr virus-transformed lymphoblastoid cell lines.

Epstein-Barr virus-transformed human B lymphoblastoid cell lines (EBV-LCL) can present soluble antigens to antigen-primed T lymphocytes. In this study, we used HLA antigen-loss mutants of an EBV-LCL line (LCL 721) to demonstrate that the presentation of a soluble antigen from Candida albicans (CAN) by EBV-LCL to primed T cells can be restricted by multiple HLA determinants. Haplotype-deletion mutants that contained only the maternal or only the paternal HLA-haplotype were used to demonstrate the preferential role of autologous HLA antigens in presenting soluble antigens to Candida-primed T cells from the donor of LCL-721, and to T cells from her mother and father. Immunoselected mutants of LCL-721 showing a variety of distinct phenotypes that are deficient in HLA-DR, DQ, or DP antigen expression were tested as antigen-presenting cells. The antigen-presenting ability of these class II deficient EBV-LCL variants weakened with progressive loss of class II HLA determinants expressed on the cell surface. Our study, therefore, provides evidence for multiple HLA restriction determinants, including HLA-DR, DQ, and DP. Furthermore, LCL lacking all HLA-DR, DQ, and DP expression because of homozygous deletion of these MHC class II genes still presented CAN and Tetanus toxid (TET), although to a much lesser degree than presented by LCL-721. This suggests that determinants other than DR, DQ, and DP which are expressed on these EBV-LCL may also function as restriction elements for the proliferative T-cell response to soluble antigens.