Activation of an alternative death receptor-induced signaling pathway in human hepatocytes under caspase arrest

Caspases are thought to be essential in execution of death receptor-induced apoptosis. However, recent findings suggest the existence of alternative pathways independent of caspases. We provide further evidence for such signaling in hepatocytes. RESULTS: Death receptor-induced activation of caspases and apoptosis in primary murine hepatocytes was completely blocked in presence of 1.5 microM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk). Whereas the same concentration of the inhibitor was sufficient to block TNF receptor 1-, CD95- or TRAIL receptor 1/-2-induced activation of caspases in primary human hepatocytes or HepG2 cells, complete prevention apoptotic cell death needed almost 100 microM zVAD-fmk. Under caspase-inhibitory but non-protective conditions, i.e. at 1.5 microM zVAD-fmk, various serine protease inhibitors prevented apoptosis-like cell death. Neither sole arrest of caspases nor inhibition of serine proteases alone protected human hepatocytes. CONCLUSION: Human but not murine hepatocytes bear the potential to activate a permissive, serine protease inhibitor-sensitive alternative death signaling pathway under caspase-inhibitory conditions.     

Reference values for height, weight and body mass index of German born Turkish children

Turkish children and adolescents born in Northern Europe grow different from native Northern European children, but reference values for height, weight and BMI for these children do not exist. With this study, we intend to provide growth standards for German born Turkish children. Data were obtained from 797 Turkish children and adolescents born in Germany age 0-25.8 years (males), respectively 0-18.3 years (females). We generated synthetic reference values for height, weight, and BMI. The results show that Turkish children and adolescents are heavier after the age of 6 years, and that they remain short after puberty. Eighteen year old Turkish men, and 15-year-old Turkish women are shorter (males 175.2 cm vs. 180.4 cm, p < 0.05; females 159.3 cm vs. 165.0 cm, p < 0.05), and heavier than Germans. Six out of 53 young Turkish men and 9 out of 100 young Turkish women were obese. Twelve out of 53 young Turkish men (23%) and 18 out of 100 young Turkish women (18%) have fallen below the 3rd centile for height. It can be concluded that growth of Turkish children and adolescents born in Germany significantly differs from native children. Reference LMS values for body height, weight and BMI of German born Turkish boys and girls are presented.     

Model for the regulation of size in the wing imaginal disc of Drosophila

For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. Here, we present a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations we show that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc.     

Distinct functions of alpha-Spectrin and beta-Spectrin during axonal pathfinding

Cell-shape changes during development require a precise coupling of the cytoskeleton with proteins situated in the plasma membrane. Important elements controlling the shape of cells are the Spectrin proteins that are expressed as a subcortical cytoskeletal meshwork linking specific membrane receptors with F-actin fibers. Here, we demonstrate that Drosophila karussell mutations affect beta-spectrin and lead to distinct axonal patterning defects in the embryonic CNS. karussell mutants display a slit-sensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals. Cell-type-specific rescue experiments demonstrate that beta-Spectrin is required autonomously and non-autonomously in cortical neurons to allow normal axonal patterning. Within the cell, beta-Spectrin is associated with alpha-Spectrin. We show that expression of the two genes is tightly regulated by post-translational mechanisms. Loss of beta-Spectrin significantly reduces levels of neuronal alpha-Spectrin expression, whereas gain of beta-Spectrin leads to an increase in alpha-Spectrin protein expression. Because the loss of alpha-spectrin does not result in an embryonic nervous system phenotype, beta-Spectrin appears to act at least partially independent of alpha-Spectrin to control axonal patterning.     

Suture Closure versus Non-Closure of Subcutaneous Fat and Cosmetic Outcome after Cesarean Section: A Randomized Controlled Trial

Introduction

To investigate the effect of subcutaneous fat suture closure versus non-closure at cesarean section (CS) on long-term cosmetic outcome.

Material and Methods

Women undergoing planned or unplanned CS were randomized to either subcutaneous fat suture closure or non-closure using a 1∶1 allocation algorithm. Participants and outcome assessors were blinded to group allocation. Scar evaluation was performed after two and six months. Primary outcome measures were Patient and Observer Scar Assessment Scale (POSAS) summary scores six months after surgery. Secondary outcome measures were Vancouver Scar Scale (VSS) summary scores, retraction of the scar below the level of the surrounding skin, duration of surgery, and development of hematoma, seroma, surgical site infection (SSI) or wound disruption. Data were analyzed according to the intention to treat principle.

Results

A total of 116 women were randomized and 91 participants, 47 in the closure and 44 in the non-closure group, completed the trial and were analyzed. There were no differences in patient morphometrics or surgery indications between groups. At two and six months no significant differences were found with respect to POSAS or VSS scores between groups. After two months significantly more women in the non-closure group described their scar as being retracted below the level of the skin (36% vs. 15%, p = 0.02) whereas no difference was observed at six months. There were significantly more hematomas in the non-closure (25%) compared to the closure group (4%) (p = 0.005). There was no difference in duration of surgery, SSI, seroma formation or wound disruption between groups.

Conclusions

Suture closure of the subcutaneous fat at CS does not affect long-term cosmetic outcome. (Level I evidence).

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01542346","term_id":"NCT01542346"}}NCT01542346.     

Cationic Synthetic Peptides: Assessment of Their Antimicrobial Potency in Liquid Preserved Boar Semen

Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.     

Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.     

Liver Sinusoidal Endothelial Cells Are a Site of Murine Cytomegalovirus Latency and Reactivation

Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients'' tissues. The viral genome was found to localize primarily to sry-negative CD11b⁻ CD11c⁻ CD31⁺ CD146⁺ cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146⁺ cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10⁴ LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients'' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.In human cytomegalovirus (hCMV) infection, hematopoietic progenitor cells of the myeloid differentiation lineage are a recognized cellular site of virus latency (for more-recent reviews, see references 75 and 94), and cell differentiation-dependent as well as cytokine-mediated viral gene desilencing by chromatin remodeling is discussed as the triggering event leading to virus reactivation (for a review, see reference 7). Although hematopoietic stem cell or bone marrow transplantation (BMT) is frequently associated with hCMV reactivation and recurrence in recipients after hematoablative leukemia/lymphoma therapy, the incidence of virus recurrence and disease is highest in the combination of an hCMV-negative donor (D⁻) and an hCMV-positive recipient (R⁺) (D⁻R⁺ > D⁺R⁺ > D⁺R⁻), indicating that donor hematopoietic cells are not the only source of latent hCMV and actually not the predominant source (34). Rather, the recipients experience reactivation of their own virus. Just the opposite is true in the case of solid organ transplantation, where the reactivating virus is mostly transmitted with the transplanted organ (D⁺R⁻ > D⁺R⁺ > D⁻R⁺) (34). Collectively, these risk assessments support the suggestion that reactivation, in both instances, occurs in latently infected tissue cells, that is, within the recipient''s organs and the transplanted donor organ, respectively. Although tissue-resident cells of hematopoietic origin remain candidates, stromal and parenchymal tissue cells come into consideration as additional sites of CMV latency.Longitudinal analysis of viral genome load in the latency models of murine CMV (mCMV) infection of neonatal mice (9, 91) as well as of adult mice after experimental BMT (8, 62, 64) has demonstrated a high viral latency burden in multiple organs long after clearance of viral DNA from bone marrow and blood (reviewed in reference 92). These findings support the suggestion that there exist two types of latency, namely, a temporary latency in hematopoietic cells and a latency in tissue cells that lasts through life. Accordingly, both types of latency may coexist early after primary infection, while “late latency” is restricted to organ sites. As we have shown previously in a sex-mismatched murine BMT model, bone marrow cells (BMCs) derived from latently infected donors in the phase of organ-restricted “late latency” cannot transmit latent or reactivated infection to naïve recipients upon intravenous cell transfer (99).A first hint for mCMV latency in stromal or reticular cells was presented long ago by Mercer and colleagues (73), who showed that infected cells during acute infection of the spleen are predominantly sinusoidal lining cells and that latent mCMV can be recovered from a major histocompatibility complex class II (MHC-II) antigen-negative and Thy-1 (CD90)-negative “stromal” cell fraction, which includes endothelial cells (ECs). These findings strongly argued against T and B lymphocytes, macrophages, and dendritic cells (DCs) being major reservoirs of latent mCMV in the spleen, a conclusion supported by later work of Pomeroy and colleagues (86). Similarly, Klotman and colleagues (54) as well as Hamilton and Seaworth (44) concluded that in kidney transplantation, donor kidney is the source of latent mCMV and that the latent viral genome is harbored by renal peritubular epithelial cells (53). A first hint for mCMV latency in ECs within the liver was provided by in situ PCR images presented by Koffron and colleagues (59) showing nuclear staining in cells with a microanatomical localization suspicious of liver sinusoidal ECs (LSECs). For hCMV, ECs, in particular those in arterial vessel walls, are regarded as a site of latency on the basis of the presence of viral DNA in cells expressing an EC marker (81; for a review, see reference 48), although other authors did not detect viral DNA in venous vessel walls (95). As discussed by Jarvis and Nelson (48), these data are not necessarily conflicting but may rather reflect the diversity of EC subsets at different anatomical locations (21, 27). As far as we know, and reactivation of productive infection from ECs that carry latent viral DNA is not yet formally proven for any type of EC.Hepatitis is a relevant organ manifestation of CMV disease in immunocompromised hosts (65), and hCMV reactivation has been reported to be the most common cause of infection following liver transplantation, in particular in a D⁺R⁻ combination (34, 76). In the murine model of immunocompromised hosts, viral histopathology in the liver is dominated by the cytopathogenic infection of hepatocytes, leading to extended plaque-like tissue lesions (41, 84; reviewed in reference 45). Occasionally, however, in these studies, infected hepatic ECs as well as Kupffer macrophages were detected by virus-specific immunohistology or by in situ virus-specific DNA hybridization.Using cell-type-specific conditional recombination of a fluorescence-tagged reporter virus in Cre-transgenic mice expressing Cre selectively in hepatocytes under the control of the albumin promoter, hepatocytes were recently identified as the main virus-producing cell type during mCMV infection. In Cre-transgenic mice expressing Cre selectively in vascular ECs under the control of the Tie2 promoter, the reporter virus was found to recombine also in LSECs, which released an amount of virus sufficient for virus spread to neighboring hepatocytes, although the virus productivity of LSECs was low and contributed little to the overall virus load in the liver (97).LSECs represent a unique liver-resident population of antigen-presenting cells that bear the capacity to cross-present antigens to naïve CD8 T cells (68, 69; reviewed in reference 57). They constitute the fenestrated endothelial lining of the hepatic sinusoids (15). By separating the sinusoidal compartment of the liver from the space of Disse and the liver parenchyma, LSECs form a boundary surface for sensing of pathogens and interaction with passenger lymphocytes. They perform a scavenger function contributing to hepatic clearance of bacterial degradation products derived from the gastrointestinal tract (103; reviewed in reference 57). According to this physiological role, antigen presentation by LSECs is associated with tolerance induction rather than with triggering an inflammatory immune response (29, 56, 68, 104).Here we provide evidence to support the suggestion that mCMV has chosen the tolerogenic and long-lived LSECs as an immunoprivileged niche for establishing viral latency in the liver.     

Sticky connections: extracellular matrix protein recognition and integrin-mediated cellular invasion by Staphylococcus aureus

Staphylococcus aureus is a leading cause of hospital-acquired and often persistent infections. A key feature of pathogenic S. aureus is the expression of an array of extracellular matrix-binding proteins. In particular, the fibronectin-binding proteins FnBP-A and FnBP-B afford the pathogen the ability to connect to cellular integrins and to trigger internalization into host cells. Recent work has highlighted the role of host cell invasion in the pathogenesis of S. aureus, the structure-function relationship of FnBPs, and the host factors required to allow bacterial uptake. Understanding the invasive capacity of S. aureus should open up new avenues to control this microorganism in diverse disease settings.     

A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b

Gamma-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor alpha (TNFalpha) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.