Direct Heme Transfer Reactions in the Group A Streptococcus Heme Acquisition Pathway

The heme acquisition machinery in Group A Streptococcus (GAS) consists of the surface proteins Shr and Shp and ATP-binding cassette transporter HtsABC. Shp cannot directly acquire heme from methemoglobin (metHb) but directly transfers its heme to HtsA. It has not been previously determined whether Shr directly relays heme from metHb to Shp. Thus, the complete pathway for heme acquisition from metHb by the GAS heme acquisition machinery has remained unclear. In this study, the metHb-to-Shr and Shr-to-Shp heme transfer reactions were characterized by spectroscopy, kinetics and protein-protein interaction analyses. Heme is efficiently transferred from the β and α subunits of metHb to Shr with rates that are 7 and 60 times greater than those of the passive heme release from metHb, indicating that Shr directly acquires heme from metHb. The rapid heme transfer from Shr to Shp involves an initial heme donor/acceptor complex and a spectrally and kinetically detectable transfer intermediate, implying that heme is directly channeled from Shr to Shp. The present results show that Shr speeds up heme transfer from metHb to Shp, whereas Shp speeds up heme transfer from Shr to HtsA. Furthermore, the findings demonstrate that Shr can interact with metHb and Shp but not HtsA. Taken together with our published results on the Shp/HtsA reaction, these findings establish a model of the heme acquisition pathway in GAS in which Shr directly extracts heme from metHb and Shp relays it from Shr to HtsA

Extensive Regulation of Diurnal Transcription and Metabolism by Glucocorticoids.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism

Additive Anti-Tumor Effects of Lovastatin and Everolimus In Vitro through Simultaneous Inhibition of Signaling Pathways

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis work was supported by a research grant from the Ludwig-Maximilians University of Munich (Förderprogramm für Forschung und Lehre [FöFoLe], grant number 865/829)

Patients with femoral or distal forearm fracture in Germany: a prospective observational study on health care situation and outcome

BACKGROUND: Distal radius and proximal femoral fractures are typical injuries in later life, predominantly due to simple falls, but modulated by other relevant factors such as osteoporosis. Fracture incidence rates rise with age. Because of the growing proportion of elderly people in Western industrialized societies, the number of these fractures can be expected to increase further in the coming years, and with it the burden on healthcare resources. Our study therefore assessed the effects of these injuries on the health status of older people over time. The purpose of this paper is to describe the study method, clinical parameters of fracture patients during hospitalization, mortality up to one and a half years after discharge in relation to various factors such as type of fracture, and to describe changes in mobility and living situation. METHODS: Data were collected from all consecutive patients (no age limit) admitted to 423 hospitals throughout Germany with distal radius or femoral fractures (57% acute-care, femoral and forearm fractures; 43% rehabilitation, femoral fractures only) between January 2002 and September 2003. Polytrauma and coma patients were excluded. Demographic characteristics, exact fracture location, mobility and living situation, clinical and laboratory parameters were examined. Current health status was assessed in telephone interviews conducted on average 6–7 months after discharge. Where telephone contact could not be established, at least survival status (living/deceased/date of death) was determined. RESULTS: The study population consisted of 12,520 femoral fracture patients (86.8% hip fractures), average age 77.5 years, 76.5% female, and 2,031 forearm fracture patients, average age 67.6 years, 81.6% female. Women's average age was 6.6 (femoral fracture) to 10 years (forearm fracture) older than men's (p < 0.0001). Only 4.6% of femoral fracture patients experienced changes in their living situation post-discharge (53% because of the fracture event), although less than half of subjects who were able to walk without assistive devices prior to the fracture event (76.7%) could still do so at time of interview (34.9%). At time of interview, 1.5% of subjects were bed-ridden (0.2% before fracture). Forearm fracture patients reported no change in living situation at all. Of the femoral fracture patients 119 (0.95%), and of the forearm fracture patients 3 (0.15%) died during hospital stay. Post-discharge (follow-up one and a half years) 1,463 femoral fracture patients died (19.2% acute-care patients, 8.5% rehabilitation patients), but only 60 forearm fracture patients (3.0%). Ninety percent of femoral fracture deaths happened within the first year, approximately 66% within the first 6 months. More acute-care patients with a pertrochanteric fracture died within one year post-discharge (20.6%) than patients with a cervical fracture (16.1%). CONCLUSION: Mortality after proximal femoral fracture is still alarmingly high and highest after pertrochanteric fracture. Although at time of interview more than half of femoral fracture patients reported reduced mobility, most patients (96%) attempt to live at home. Since forearm fracture patients were on average 10 years younger than femoral fracture patients, forearm fractures may be a means of diagnosing an increased risk of later hip fractures

Dendritic cell-specific delivery of Flt3L by coronavirus vectors secures induction of therapeutic antitumor immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8(+) T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8(+) T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity

Acute mucosal pathogenesis of feline immunodeficiency virus is independent of viral dose in vaginally infected cats

<p>Abstract</p> <p>Background</p> <p>The mucosal pathogenesis of HIV has been shown to be an important feature of infection and disease progression. HIV-1 infection causes depletion of intestinal lamina propria CD4+ T cells (LPL), therefore, intestinal CD4+ T cell preservation may be a useful correlate of protection in evaluating vaccine candidates. Vaccine studies employing the cat/FIV and macaque/SIV models frequently use high doses of parenterally administered challenge virus to ensure high plasma viremia in control animals. However, it is unclear if loss of mucosal T cells would occur regardless of initial viral inoculum dose. The objective of this study was to determine the acute effect of viral dose on mucosal leukocytes and associated innate and adaptive immune responses.</p> <p>Results</p> <p>Cats were vaginally inoculated with a high, middle or low dose of cell-associated and cell-free FIV. PBMC, serum and plasma were assessed every two weeks with tissues assessed eight weeks following infection. We found that irrespective of mucosally administered viral dose, FIV infection was induced in all cats. However, viremia was present in only half of the cats, and viral dose was unrelated to the development of viremia. Importantly, regardless of viral dose, all cats experienced significant losses of intestinal CD4+ LPL and CD8+ intraepithelial lymphocytes (IEL). Innate immune responses by CD56+CD3- NK cells correlated with aviremia and apparent occult infection but did not protect mucosal T cells. CD4+ and CD8+ T cells in viremic cats were more likely to produce cytokines in response to Gag stimulation, whereas aviremic cats T cells tended to produce cytokines in response to Env stimulation. However, while cell-mediated immune responses in aviremic cats may have helped reduce viral replication, they could not be correlated to the levels of viremia. Robust production of anti-FIV antibodies was positively correlated with the magnitude of viremia.</p> <p>Conclusions</p> <p>Our results indicate that mucosal immune pathogenesis could be used as a rapid indicator of vaccine success or failure when combined with a physiologically relevant low dose mucosal challenge. We also show that innate immune responses may play an important role in controlling viral replication following acute mucosal infection, which has not been previously identified.</p

Circulating Endothelial Progenitor Cells in Kidney Transplant Patients

Background: Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs. Methods: We analyzed 52 RTx patients (58613 years; 33 males, mean 6 SD) and 16 age- and gender-matched subjects with normal kidney function (57617; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry. Results: Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1. Conclusions: We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.

Deep Sequencing of MYC DNA-Binding Sites in Burkitt Lymphoma

BACKGROUND: MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far. METHODOLOGY/PRINCIPAL FINDINGS: ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing. CONCLUSION/SIGNIFICANCE: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology

Whole exome re-sequencing implicates CCDC38 and cilia structure and function in resistance to smoking related airflow obstruction

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1) despite many years of heavy smoking. We # hypothesised that these ‘‘resistant smokers’’ may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the ‘‘resistant smokers’’ and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.3461024) in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke