[Childhood obesity and coronary artery disease: a Mendelian randomization study].

Objective: To assess the casual effect of childhood obesity on adulthood coronary artery disease (CAD) using Mendelian randomization (MR) method. Methods: Data on BMI of children aged 2-10 years in 2015 were downloaded from Early Growth Genetics Consortium and Genetic Investigation of Anthropometric Traits Consortium. Twenty-seven genetic variants related to children's BMI were selected as instrumental variables (IVs), and the associations between IVs and CAD were extracted from a Meta-analysis of the genome-wide association study of CAD cases published in UK Biobank 2015. We used MR-Egger regression to test whether there was the pleiotropy of the selected SNPs. In the present MR methods, we conducted MR analyses by using mode-based estimate method as primary method for summary-level of associations to estimate the causal association between childhood obesity and CAD. Results: The intercept term estimated for CAD from MR-Egger method suggested that the selected SNPs don't exert pleiotropy with a 95%CI including the null (-0.008-0.018). In addition, we found evidence that support the effect of childhood obesity on CAD risk: a 1 s increase in children BMI (kg/m(2)), and the risk of suffering from CAD in adulthood increased by an average of 37% (OR=1.37, 95%CI: 1.09-1.72). Conclusion: This study provides a causal association between childhood obesity and CAD risk.

Adiposity and blood pressure among 55 000 relatively lean rural adults in southwest of China.

This corrects the article DOI: 10.1038/jhh.2014.129.

Adiposity and blood pressure among 55 000 relatively lean rural adults in southwest of China.

Obesity is a strong determinant of blood pressure. Uncertainty remains, however, about which indices of adiposity most strongly predict blood pressure, particularly among those who were relatively lean, such as those from rural China. We analyzed cross-sectional data on 55 ,687 (38.3% men) participants aged 30-79 years who were enrolled into the China Kadoorie Biobank from a rural county in southwest of China during 2004-2008. Measured body mass index (BMI) and waist circumference (WC) were related to blood pressure in multivariable linear regression analyses. The overall mean values of BMI, WC, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were 23.3 kg m(-2), 78.0 cm, 129.2 mm Hg and 77.2 mm Hg, respectively. There was a strongly positive, and apparently linear, relationship of BMI and WC with blood pressure, with 1 s.d. higher BMI associated with 4.3/2.3 mm Hg higher SBP/DBP and 1 s.d. WC associated with 3.8/2.1 mm Hg (P<0.0001). Additional adjustment for WC only slightly attenuated the association of BMI with blood pressure, whereas additional adjustment for BMI almost completely eliminated the association of WC with blood pressure. Our findings suggest that in relatively lean Chinese adults, general adiposity is more strongly assciated with blood pressure than central adiposity.

Modified AdCTLA-4 vector blocks alloimmune response in vitro.

BACKGROUND: Gene transfer of the costimulatory blockade molecule CTLA-4Ig into cold-preserved rat liver allografts results in indefinite allograft survival. Despite local delivery, this mode of immunomodulation is associated with systemic immunosuppression. In an effort to restrict immunosuppression to the graft, we have constructed a novel adenoviral vector, AdCTLA-4ex-TAG, in which the Ig sequence of CTLA-4Ig DNA has been deleted to destabilize the gene product to promote rapid extrahepatic degradation while maintaining its immunosuppressive activity within the graft milieu. METHODS: (1) Vector construction. CTLA-4 extracellular binding domain (CTLA-4ex) was prepared by PCR-based cloning methods and fused in frame to a genetic element encoding an epitope TAG allowing identification of the transgene product CTLA-4exTAG. CTLA-4exTAG was subcloned into a shuttle vector enabling isolation of AdCTLA-4exTAG. (2) In vitro transfection: AdCTLA-4exTAG was transfected into MH(1)C(1) cells and then supernatant was recovered for Western blot analysis. (3) In vitro alloimmune response was characterized by CFSE proliferation assay. (4) Extrahepatic effect of AdCTLA-4exTAG was characterized by the ability to control tumor growth after implantation of a regressive, immune sensitive cancer cell line (REGb) in the skin of BDIX rats after liver transduction with AdCTLA-4exTAG. RESULTS: Expression and secretion of the recombinant protein were documented by Western blot after infection of the MH(1)C(1) cell line() with AdCTLA-4exTAG. Addition of infected MH(1)C(1) cell supernatant resulted in abrogation of alloimmune response as shown by markedly diminished division of CD4(+) T cells in a CFSE proliferation assay. Extrahepatic tumor regressed normally after liver transduction with AdCTLA-4exTAG. CONCLUSIONS: These results show efficient in vitro expression of CTLA-4exTAG after transfection with AdCTLA-4exTAG. The modified protein retains its ability to abrogate in vitro alloimmune response. Efficient control of extrahepatic tumor growth after liver-directed delivery of AdCTLA-4exTAG suggests that the immunosuppressive effect of this vector is restricted to the liver. These results set the ground for the utilization of this novel adenoviral vector in the transplant setting.

Activation of interleukin-6/STAT3 and liver regeneration following transplantation.

BACKGROUND: Every liver that is procured, stored, and transplanted experiences injury from cold ischemia and reperfusion. Most recover quickly, but some grafts sustain enough injury to result in prolonged organ dysfunction or require retransplantation. The molecular mechanisms involved in early graft function and recovery following cold ischemia and reperfusion (I/R) after liver transplantation have not been well defined. Interleukin (IL)-6 is a critical factor in the mitogenic response within the liver, and is important for cell cycle progression and protection from injury. Activation of the latent transcription factor, STAT3, is dependent on IL-6 release. The role of the IL-6/STAT3 pathway and hepatocellular regeneration in graft recovery and cell cycle progression following cold ischemia and reperfusion was studied in a rat liver transplant orthotopic (OLT) model. Methods. Rat OLT was performed in a syngeneic model. The presence, time course, and magnitude of expression of IL-6, STAT3 activation, and upregulation of target immediate early genes were determined in liver grafts with minimal (<1 h) and prolonged (12 h) cold preservation times followed by transplantation. Progression of the cell cycle and replication was confirmed by BrdU uptake. RESULTS: Prolonged cold ischemia resulted in increased IL-6 expression and STAT3 activation. This correlated with upregulation of junB, c-fos, c-myc, and c-jun, immediate early genes associated with hepatic regeneration. Extensive DNA replication was present in livers with 12-h ischemia, demonstrating successful completion of the cell cycle. CONCLUSIONS: The participation of the IL-6/STAT3 pathway leading to cell cycle progression and regeneration is an important component in the recovery of organs immediately following cold preservation and transplantation.

The role of alphabeta- and gammadelta-T cells in allogenic donor marrow on engraftment, chimerism, and graft-versus-host disease.

BACKGROUND: We previously characterized a facilitating cell (FC) in mouse marrow that enables engraftment of allogeneic hematopoietic stem cells (HSCs) without causing graft-versus-host disease (GVHD). The FC shares some cell surface molecules with T cells (Thy1+, CD3epsilon+, CD8+, CD5+, and CD2+) but is T-cell receptor (TCR) negative. Historically, depletion of CD3+ or CD8+ cells from rat marrow was associated with an increased rate of failure of engraftment. In this study, we evaluated whether depletion of alphabeta- and gammadelta-TCR(+) T cells from donor marrow would retain engraftment potential yet avoid GVHD. METHODS: Wistar-Furth rats were conditioned with 950 cGy of total body irradiation and transplanted with ACI bone marrow processed to remove either alphabeta-TCR(+), gammadelta-TCR(+), or alphabeta- plus gammadelta-TCR(+) T cells. Recipients were typed for chimerism at 28 days and monthly thereafter. RESULTS: Recipients of marrow depleted of alphabeta- (group A), gammadelta- (group B), or alphabeta- and gammadelta-TCR(+) T cells (group C) engrafted and had an average chimerism level of 73.0+/-8.3%, 92.3+/-9.2%, and 46.3+/-32.8%, respectively. Aggressive T-cell depletion did not remove the FC population (CD8+/CD3+/TCR(-)). Group A and group B both developed GVHD, with a higher incidence of GVHD in group B compared to group A. None of the recipients in group C developed GVHD. CONCLUSIONS: These data demonstrate that depletion of T cells from rat marrow does not impair engraftment of HSCs, indirectly supporting the existence of FCs in rat marrow. Moreover, donor alphabeta- and gammadelta-TCR(+) T cells contribute to GVHD in a nonredundant fashion, although alphabeta-TCR(+) T cells are more potent as the effector cells. Finally, the level of donor chimerism is influenced by the composition of the graft, because recipients of marrow that contain alphabeta-TCR(+) T cells exhibited significantly higher donor chimerism compared to recipients of marrow depleted of both alphabeta- and gammadelta-TCR(+) T cells.

Two genes encoding unique proliferating-cell-nuclear-antigens are expressed in Toxoplasma gondii.

Complete cDNA sequences encoding two novel proliferating-cell-nuclear-antigens (designated TgPCNA1 and 2) were isolated from a Toxoplasma gondii tachyzoite cDNA library, and Southern analysis using cDNA probes confirmed the presence of two PCNA genes in T. gondii genomic DNA. Expressed-sequence-tags were identified in the T. gondii database that matched each TgPCNA cDNA and closely related PCNA coding regions (designated PfPCNA1 and 2) were discovered in sequence data obtained from chromosome 12 and 13 of Plasmodium falciparum. TgPCNA1 and PfPCNA1 were found to share the highest amino acid identity at 49% compared to TgPCNA2 and PfPCNA2 (37% identity) whereas intraspecies PCNAs were determined to be less similar (27-30% identity). Phylogenetic analysis suggests the two apicomplexan PCNAs are the result of a gene duplication in the common ancestor of these parasites. Antibodies specific for TgPCNA1 ( approximately 40 kDa) or TgPCNA2 ( approximately 37 kDa) detected single antigen species in tachyzoite extracts that were expressed at similar levels in isolates representative of the T. gondii Type I, II and III strains. TgPCNA1-specific cDNA probes detected multiple mRNA species on Northern blots, which when combined, were expressed 5-7 fold higher than the single species of mRNA detected by the TgPCNA2 probe. The difference in the number of mRNA species and comparative mRNA levels suggests each TgPCNA gene is independently controlled, although in light of the nearly equal levels of protein a post-transcriptional mechanism may be responsible for equalizing protein expression.

Cysteine proteinases and the pathogenesis of amebiasis.

Amebiasis is a major cause of morbidity and mortality throughout the tropical world. Entamoeba histolytica is now recognized as a separate species from the morphologically identical E. dispar, which cannot invade. Cysteine proteinases are a key virulence factor of E. histolytica and play a role in intestinal invasion by degrading the extracellular matrix and circumventing the host immune response through cleavage of secretory immunoglobulin A (sIgA), IgG, and activation of complement. Cysteine proteinases are encoded by at least seven genes, several of which are found in E. histolytica but not E. dispar. A number of new animal models, including the formation of liver abscesses in SCID mice and intestinal infection in human intestinal xenografts, have proven useful to confirm the critical role of cysteine proteinases in invasion. Detailed structural analysis of cysteine proteinases should provide further insights into their biochemical function and may facilitate the design of specific inhibitors which could be used as potential chemotherapeutic agents in the future.

Alpha beta TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice.

BACKGROUND: Although the transplantation of solid organs and cellular grafts is a clinical routine, the morbidity and mortality associated with immunosuppression is significant. This could be avoided by the induction of donor-specific tolerance. To develop targeted antirejection strategies and regimens to induce donor-specific tolerance, cell populations in the recipient-mediating rejection of solid organ and cellular grafts must be defined. In this study we examined the role of alpha beta-TCR+ cells in the rejection of allogeneic heart grafts, by use of knockout (KO) mice deficient in the production of alpha beta-TCR+ T cells. METHODS: C57BL/6-TcrbtmlMom (alpha beta-KO) and C57BL6/J (B6) recipient mice were transplanted with B10.BR/SgSnJ (B10.BR) or BALB/c heart allografts. Animals also received bone marrow from normal B10.BR donors, followed by donor-specific or third-party heart transplants. RESULTS: Naive B6 control mice rejected B10.BR and BALB/c grafts within 16 days. In striking contrast, B10.BR and BALB/c heart allografts were indefinitely accepted in unmanipulated alpha beta-KO mice. The immune responsiveness was restored after bone marrow transplantation from normal donors. After bone marrow transplantation major histocompatibility-disparate BALB/c third-party heart grafts were rejected, whereas donor-specific grafts were still accepted. CONCLUSIONS: alpha beta-TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice. Bone marrow chimerism is associated with donor-specific transplantation tolerance.

Pantropic retroviral vectors mediate gene transfer and expression in Entamoeba histolytica.

Transformation of Entamoeba histolytica has been previously reported, but the foreign genes have all been replicated episomally. Pantropic retroviral vectors based on the Moloney murine leukemia virus with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) have an extremely broad host range and can be concentrated to high titer. To investigate whether these pseudotyped, pantropic vectors can mediate gene transfer and expression in E. histolytica, we constructed a retroviral vector, in which a hygromycin phosphotransferase is expressed from the E. histolytica actin promoter. Data confirm the infection, integration, and expression of a foreign gene mediated by the provirus. To our knowledge, this is the most evolutionarily distant example of successful integration and expression of a mammalian retrovirus. Pantropic retroviral vectors may thus facilitate genetic analysis in species lacking transformation systems.

Cellular and transcriptional changes during excystation of Giardia lamblia in vitro.

Excystation of Giardia lamblia entails differentiation of dormant cysts into parasitic trophozoites. Despite its importance for infection, this transformation is not understood at the cellular or molecular levels. In these studies, we report that excystation entails detection of environmental stimuli across the tough extracellular cyst wall leading to highly coordinated physiological, structural, and molecular responses. We found that novel cytoplasmic rearrangements and changes in individual species of mRNA and in cytoplasmic pH occur within the cyst wall in the earliest stage of excystation, in response to conditions modeling cyst ingestion and passage into the human stomach. This suggests that cysts do not contain all the mRNA needed for excystation and emergence and supports our hypothesis that external stimuli, including hydrogen ions, may penetrate or be perceived across the cyst wall. In contrast, changes in cyst wall structure or proteins were detected only later in excystation, in the stage that models passage into the human small intestine, where trophozoites can emerge and survive. These findings show that excystation of G. lamblia is a highly complex and active process and provide important insights into its cellular and molecular components.

Suppression of intracellular hydrogen peroxide generation and catalase levels in CD8+ T-lymphocytes from HIV+ individuals.

CD8+ T-lymphocytes from HIV+ individuals contain short telomeres, a sign of cell senescence. To test our hypothesis that the cell type is functionally defective in the biochemical indices related to cell proliferation, we investigated the profiles of intracellularly generated H2O2 levels with or without PMA as well as immunoreactive catalase levels using flow cytometric method. We observed that, in HIV+ but not in HIV- individuals, the constitutively generated H2O2 level was significantly lower in CD8+ T-cells compared with CD4+ T-cells. Importantly, activated effector CD8+CD28- cells showed remarkably low H2O2 levels compared with CD8+CD28+ cells, and the latter in HIV+ individuals also showed low levels. A similar defect of CD8+ cells of HIV+ individuals was also seen with H2O2 levels stimulated with PMA in the presence of a catalase inhibitor. Furthermore, the immunoreactive catalase content was lower in CD8+ cells compared with CD4+ cells only in HIV+ individuals. These results suggest that CD8+ T-lymphocytes are functionally defective with the constitutively generated and PMA-elicited levels of H2O2 and the corresponding scavenger. Diminished immunocompetence of HIV+ individuals may be caused, in part, by the functional defect of CD8+ T-cells.

The role of extracellular cysteine proteinases in pathogenesis of Entamoeba histolytica invasion.

The extracellular cysteine proteinases of Entamoeba histolytica have been implicated as important virulence factors in the pathogenesis of amebiasis and play a key role in tissue invasion and disruption of host defenses. These proteinases have attracted considerable interest as targets for novel therapeutic agents and as vaccine candidates. Here, Xuchu Que and Sharon Reed highlight some of the more recent findings, focusing in particular on functional and structural features of the extracellular cysteine proteinases of E. histolytica.

Developmentally regulated transcripts and evidence of differential mRNA processing in Giardia lamblia.

Although encystation and excystation are crucial to transmission of Giardia lamblia, little is known about the regulation of these very distinct differentiation processes. Fingerprinting of giardial mRNA populations throughout the time course of differentiation demonstrated complex patterns in mRNA differential display. Certain transcripts appeared or increased, while others decreased or disappeared at specific times, in response to physiologic stimuli that mimic key stages in parasite descent through the host gastrointestinal tract. This approach has allowed the direct identification of critical stages in differentiation, as well as isolation of genes which may be crucial to the development of G. lamblia. One stage-specific single copy gene (ENC6) whose transcript is greatly upregulated during encystation was analyzed further. Partial sequence analysis revealed no correspondence with known genes. 3'-rapid amplification of cDNA ends (3'-RACE) analysis of ENC6 transcripts at various times of encystation revealed two polyadenylation sites. The more proximal site, 10 nucleotides past the single classic AGTAAA sequence, was utilized only during encystation and its transcript increased approximately 16-fold during the first 24 h of encystation. In contrast, a slightly divergent polyadenylation site 288 nucleotides downstream from the open reading frame (ORF) was used during both vegetative growth and encystation, although its transcript was present at low levels. These studies are the first evidence of differential mRNA processing in G. lamblia and suggest a potential role of the 3'-untranslated region (3'-UTR) in modulating gene expression during differentiation of this primitive eukaryote.

[Three-dimensional echocardiography in evaluation of left ventricular systolic function after mitral valve replacement for chronic mitral diseases with preservation of mitral apparatus].

OBJECTIVE: To assess the global left ventricular performance in 24 normal subjects, 24 patients suffered from mitral valve disease (MVD) with mitral valve replacement (MVR), and 20 patients with mitral valve replacement with preservation of mitral apparatus (MVRP). METHODS: 3DE was used in comparion with the findings of radiography (RNA), two-dimensional echocardiography (2DE), and m-mode (MME). RESULTS: EF in 10 patients with MVD as compared with RNA, 3DE showed a higher connection coefficient than 2DE and MME. The EF estimated by MME and 2DE showed no difference between NS and MVD groups, there were significant differences when compared with those examined by 3DE. Two, three months after opertion, the VED and VES in group of MVRP were significantly lower than those in group of MVR (P < 0.01), the EF of MVRP group was remarkably higher than that of MVR group (P < 0.05). CONCLUSION: 3DE has the ability to estimate the EF of left ventricle more accurately than 2DE and MMe, especially in the patients suffered from chronic MVD with LV shape deformation. The MVR with preservation of mitral apparatus for chronic MVD is beneficial to maintaining the global systolic function after surgery.

Expression and characterization of a rac family protein kinase of Entamoeba histolytica.

We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs". To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli. The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a Km of approx. 14 microM. Histone H3 and kemptide were phosphorylated at about half the rate of histone H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme. The protein kinase activity was higher in the presence of Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM. Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica.

Molecular cloning of a rac family protein kinase and identification of a serine/threonine protein kinase gene family of Entamoeba histolytica.

Eleven Entamoeba histolytica protein-serine/threonine-kinase gene segments were identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers to conserved amino acids in subdomains VI and VIII of the catalytic domain of protein-serine/threonine kinases. These ameba gene segments were homologous to myosin light chain kinases, protein kinase C, phosphorylase b kinase, and kinases that regulate glucose repression in yeast and cell growth in mammalian cells. One of these PCR products, which was homologous to the Dictyostelium discoideum protein kinase 2, was used to identify a full-length protein-serine/threonine-kinase gene (Eh rac1) from an E. histolytica genomic library. The open reading frame of Eh rac1 was 409 amino acids long (encoding a 47-kDa protein) and included an amino terminal segment containing 87 mostly charged and polar amino acids and a 322-amino acid carboxyl terminal segment containing the catalytic domain. The catalytic domain of Eh rac1 was homologous to the rac family of protein-serine/threonine-kinases, which are related to cAMP-dependent protein kinases and protein kinase Cs. Southern blots of ameba DNA showed that the Eh rac1 gene was present as a single copy in all strains tested, however pathogenic amebae expressed four times more Eh rac1 mRNAs than did nonpathogenic amebae. These studies suggest that E. histolytica, a primitive unicellular eukaryote, has a complex protein kinase family.