Two-component laser velocimeter measurements downstream of heart valve prostheses in pulsatile flow

Elevated turbulent shear stresses resulting from disturbed blood flow through prosthetic heart valves can cause damage to red blood cells and platelets. The purpose of this study was to measure the turbulent shear stresses occurring downstream of aortic prosthetic valves during in-vitro pulsatile flow. By matching the indices of refraction of the blood analog fluid and model aorta, correlated, simultaneous two-component laser velocimeter measurements of the axial and radial velocity components were made immediately downstream of two aortic prosthetic valves. Velocity data were ensemble averaged over 200 or more cycles for a 15-ms window opened at peak systolic flow. The systolic duration for cardiac flows of 8.4 L/min was 200 ms. Ensemble-averaged total shear stress levels of 2820 dynes/cm2 and 2070 dynes/cm2 were found downstream of a trileaflet valve and a tilting disk valve, respectively. These shear stress levels decreased with axial distance downstream much faster for the tilting disk valve than for the trileaflet valve.     

Population dynamics of an introduced bacterium degrading chlorinated benzenes in a soil column and in sewage sludge

The capacity of the -Proteobacterium Pseudomonas sp. strain P51, which degrades chlorinated benzenes, to metabolize 1,2,4-trichlorobenzene (TCB) under environmental conditions was tested by its release into two experimental systems. The first system consisted of laboratory scale microcosms which were operated with and without the addition of TCB and which were inoculated with sludge from a wastewater treatment plant. The second system consisted of a non sterile, water saturated soil column. We determined survival of strain P51 after its introduction and its ability to degrade TCB. The population dynamics was followed by selective plating and applying the polymerase chain reaction (PCR) to detect strain P51 and the chlorobenzene ( tcb) genes on catabolic plasmid pP51. The results showed a completely different behaviour of strain P51 in the two habitats under the applied conditions. In the soil column the P51 bacteria inoculated the entire area and their population reached 2 × 10⁶ cells/g soil. The population remained active since TCB was degraded to concentrations below the detection limit of 30 g/l. In the sludge microcosms, the number of strain P51 cells immediately decreased from 4 × 10⁷ cells/ml to 10⁵ cells/ml over a period of 2 days after inoculation, and then the strain disappeared to levels below our detection limit (10³–10⁴ cells/ml). In the reactor without TCB the population of P51 maintained a stable value of 10⁵ cells/ml during 8 days but then also decreased to levels below the detection limit. In addition, no significant TCB degradation was found in the sludge reactors. The influence of presence of TCB on maintenance of strain P51 in the two habitats is discussed. This work demonstrates the possibility to successfully apply preselected strains to degrade otherwise poorly degradable substances in complex mixed microbial communities. However, survival and activity may depend strongly on the type of system into which the strain is introduced.     

Structure of the HCMV UL16-MICB Complex Elucidates Select Binding of a Viral Immunoevasin to Diverse NKG2D Ligands

The activating immunoreceptor NKG2D promotes elimination of infected or malignant cells by cytotoxic lymphocytes through engagement of stress-induced MHC class I-related ligands. The human cytomegalovirus (HCMV)-encoded immunoevasin UL16 subverts NKG2D-mediated immune responses by retaining a select group of diverse NKG2D ligands inside the cell. We report here the crystal structure of UL16 in complex with the NKG2D ligand MICB at 1.8 Å resolution, revealing the molecular basis for the promiscuous, but highly selective, binding of UL16 to unrelated NKG2D ligands. The immunoglobulin-like UL16 protein utilizes a three-stranded β-sheet to engage the α-helical surface of the MHC class I-like MICB platform domain. Intriguingly, residues at the center of this β-sheet mimic a central binding motif employed by the structurally unrelated C-type lectin-like NKG2D to facilitate engagement of diverse NKG2D ligands. Using surface plasmon resonance, we find that UL16 binds MICB, ULBP1, and ULBP2 with similar affinities that lie in the nanomolar range (12–66 nM). The ability of UL16 to bind its ligands depends critically on the presence of a glutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) at position 169. An arginine residue at this position however, as found for example in MICA or ULBP3, would cause steric clashes with UL16 residues. The inability of UL16 to bind MICA and ULBP3 can therefore be attributed to single substitutions at key NKG2D ligand locations. This indicates that selective pressure exerted by viral immunoevasins such as UL16 contributed to the diversification of NKG2D ligands.     

The polymorphic HCMV glycoprotein UL20 is targeted for lysosomal degradation by multiple cytoplasmic dileucine motifs

Human cytomegalovirus (HCMV) is a widespread and persistent beta-herpesvirus. The large DNA genome of HCMV encodes many proteins that are non-essential for viral replication including numerous proteins subverting host immunosurveillance. One of them is the barely characterized UL20, which is encoded adjacent to the well-defined immunoevasins UL16 and UL18. UL20 is a type I transmembrane glycoprotein with an immunoglobulin-like ectodomain that is highly polymorphic among HCMV strains. Here, we show that the homodimeric UL20, by virtue of its cytoplasmic domain, does not reach the cell surface but is targeted to endosomes and lysosomes. Accordingly, UL20 exhibits a short half-life because of rapid lysosomal degradation. Trafficking of UL20 to lysosomes is determined by several, independently functioning dileucine-based sorting motifs in the cytoplasmic domain of UL20 and involves the adaptor protein (AP) complex AP-1. Combined substitution of three dileucine motifs allowed strong cell surface expression of UL20 comparable to UL20 mutants lacking the cytoplasmic tail. Finally, we show that the intracellularly located UL20 also is subject to lysosomal degradation in the context of viral infection. Altogether, from these data, we hypothesize that UL20 is destined to efficiently sequester yet-to-be defined cellular proteins for degradation in lysosomes.     

Analysis of the gut microbiota in the old order amish and its relation to the metabolic syndrome

Obesity has been linked to the human gut microbiota; however, the contribution of gut bacterial species to the obese phenotype remains controversial because of conflicting results from studies in different populations. To explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications, we studied men and women over a range of body mass indices from the Old Order Amish sect, a culturally homogeneous Caucasian population of Central European ancestry. We characterized the gut microbiota in 310 subjects by deep pyrosequencing of bar-coded PCR amplicons from the V1-V3 region of the 16S rRNA gene. Three communities of interacting bacteria were identified in the gut microbiota, analogous to previously identified gut enterotypes. Neither BMI nor any metabolic syndrome trait was associated with a particular gut community. Network analysis identified twenty-two bacterial species and four OTUs that were either positively or inversely correlated with metabolic syndrome traits, suggesting that certain members of the gut microbiota may play a role in these metabolic derangements.     

Interaction of monocytes with NK cells upon Toll-like receptor-induced expression of the NKG2D ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.     

Cytotoxic T lymphocytes show HLA-C-restricted recognition of EBV-bearing cells and allorecognition of HLA class I molecules presenting self-peptides.

Human CTL have been isolated that show self-restricted recognition of autologous lymphoblastoid cell lines and allorecognition. The lymphoblastoid cell line ligand most likely used a peptide that is expressed in EBV-bearing cells when the virus enters the lytic cycle. This peptide is presented to CD8+ CTL by HLA-Cw7 molecules. The allogeneic ligand recognized on non-EBV-infected cells is composed of a class I glycoprotein and a naturally selected self-peptide. In previous studies we demonstrated that this ligand is determined by two MHC-linked genes: one gene encodes the allogeneic class I molecule whereas the other controls the self-peptide. Despite the use of different peptides and different class I molecules, seemingly equivalent structures are formed that enable these two ligands to function as antigenic mimics of each other. CTL with the same patterns of dual specificity could be isolated from four unrelated donors, indicating that HLA-Cw7 is frequently involved in self-restricted recognition of EBV-harboring cells. Such CTL could help not only to contain lytic virus during a primary infection but also may be maintained life-long to eliminate cells in which reactivated virus appears.     

Soy protein influences the development of the metabolic syndrome in male obese ZDFxSHHF rats.

Previous investigations have demonstrated a marked effect of soy protein on the metabolic syndrome (MS). The purpose of this preliminary study was to identify the effects of soy-based diets on male obese ZDFxSHHF (fa/ fa-cp/?) rats. Animals were randomly assigned to one of four diets: control, casein (C); low-isoflavone (LIS) soy protein; high-isoflavone (HIS) soy protein; or casein + rosiglitazone (CR). Physiological, biochemical, and molecular parameters were determined at sacrifice. Body weight (p < 0.01) and food intake (p < 0.05) were lower in LIS-fed rodents. Rosiglitazone-treated animals had higher body weight and adiposity (p < 0.05). LIS and CR groups exhibited better glycemic control (p < 0.05), but with a limited effect in rosiglitazone-treated animals. HIS fed rats had higher glucose and triacylglyceride levels (p < 0.01), and lower plasma insulin (p < 0.01). Renal function parameters with the exception of an increase in systolic blood pressure (p < 0.05) were all suppressed in the LIS group (p < 0.01). The CR group had twofold PPARalpha and PPARgamma mRNA abundance (p < 0.01). LIS-fed animals also exhibited greater abundance of PPARgamma mRNA (p < 0.001), and nearly threefold FAS and CPT-1 mRNA levels (p < 0.05). HIS-fed rats also had higher abundance of CPT-1 mRNA, as well as a lower abundance of ACC mRNA (p < 0.05). Soy-based diets, influenced by isoflavone content and distinct from rosiglitazone, improved several metabolic parameters in obese ZDFxSHHF rats.