Iron-Fueled Life in the Continental Subsurface: Deep Mine Microbial Observatory, South Dakota, USA.

Iron-bearing minerals are key components of the Earth's crust and potentially critical energy sources for subsurface microbial life. The Deep Mine Microbial Observatory (DeMMO) is situated in a range of iron-rich lithologies, and fracture fluids here reach concentrations as high as 8.84 mg/liter. Iron cycling is likely an important process, given the high concentrations of iron in fracture fluids and detection of putative iron-cycling taxa via marker gene surveys. However, a previous metagenomic survey detected no iron cycling potential at two DeMMO localities. Here, we revisited the potential for iron cycling at DeMMO using a new metagenomic data set including all DeMMO sites and FeGenie, a new annotation pipeline that is optimized for the detection of iron cycling genes. We annotated functional genes from whole metagenomic assemblies and metagenome-assembled genomes and characterized putative iron cycling pathways and taxa in the context of local geochemical conditions and available metabolic energy estimated from thermodynamic models. We reannotated previous metagenomic data, revealing iron cycling potential that was previously missed. Across both metagenomic data sets, we found that not only is there genetic potential for iron cycling at DeMMO, but also, iron is likely an important source of energy across the system. In response to the dramatic differences we observed between annotation approaches, we recommend the use of optimized pipelines where the detection of iron cycling genes is a major goal. IMPORTANCE We investigated iron cycling potential among microbial communities inhabiting iron-rich fracture fluids to a depth of 1.5 km in the continental crust. A previous study found no iron cycling potential in the communities despite the iron-rich nature of the system. A new tool for detecting iron cycling genes was recently published, which we used on a new data set. We combined this with a number of other approaches to get a holistic view of metabolic strategies across the communities, revealing iron cycling to be an important process here. In addition, we used the tool on the data from the previous study, revealing previously missed iron cycling potential. Iron is common in continental crust; thus, our findings are likely not unique to our study site. Our new view of important metabolic strategies underscores the importance of choosing optimized tools for detecting the potential for metabolisms like iron cycling that may otherwise be missed.

Mid-term Results of Chimney and Periscope Grafts in Supra-aortic Branches in High Risk Patients.

PURPOSE: Report mid-term outcomes of thoracic endovascular aneurysm repair (TEVAR) with chimney and periscope grafts (CPG) in supra-aortic branches (SAB). METHODS: Retrospective analysis, from October 2009 to May 2014, of patients with aneurysms requiring TEVAR with zone 0/1/2 proximal landing in association with at least one CPG in the SAB. All patients were considered at high risk for conventional surgery. Peri-operative mortality and morbidity, retrograde type A dissection, maximum aortic transverse diameter (TD) and its post-operative evolution, endoleak, survival, freedom from cardiovascular re-interventions, and CPG freedom from occlusion during the follow-up were analysed. RESULTS: Forty-one patients (28.05% EuroScore II) with thoraco-abdominal aortic aneurysm (17%), arch aneurysm (39%), descending aneurysm (34%), and aneurysm extending from the arch to the visceral aorta (10%) were included. Fifteen (37%) patients were treated non-electively. Fifty-nine SABs were treated with the CPG technique: one, two, three, and four CPG were employed in 71%, 19%, 5%, and 5% of patients, respectively. The proximal landing was in zone 0 in 49% of patients, zone 1 in 17%, and zone 2 in 34%. Technical success was 95%. Peri-operative complications and neurological events were registered in six (14.6%) patients and there were 5 deaths (12%). At a median follow-up of 21.2 (mean 22, SD 18; range 0-65) months, type I/III endoleaks were registered in three (7%) cases and re-intervention in six (15%) patients. A significant aneurysm sac shrinkage (p<.001) was reported at mean follow-up and no significant aneurysm sac increase (>5 mm). The estimated 2 year survival, freedom from re-intervention, freedom from endoleak, and freedom from branch occlusion were 75%, 77%, 86%, and 96%, respectively. CONCLUSION: The chimney and periscope grafts technique was shown to be safe in aortic aneurysm disease involving the supra aortic branches, even in an emergency setting using off the shelf devices. Mid-term follow-up results in this high risk population are good, but longer follow-up is mandatory before this technique is used in intermediate-risk patients.

Inactivation of the LOX-1 pathway promotes the Golgi apparatus during cell differentiation of mural granulosa cells.

In female mammals, granulosa cells of the ovarian follicle differentiate into the corpus luteum after ovulation of the pregnable oocyte into the fallopian tube. During these differentiation processes several morphological alterations have to occur and the molecular basis is not fully understood. As an endpoint estradiol production from granulosa cells has to switch off in favor for progesterone production from the proceeding corpus luteum to sustain the developing embryo. Previously, we demonstrated that the multiligand receptor LOX-1 plays a critical role in steroid hormone synthesis of granulosa cells via intracellular calcium release from endoplasmic (ER)-dependent and ER-independent calcium pools. In the present study, we show that inhibition of LOX-1 leads to a rearrangement of ceramide from the basal membrane toward the Golgi apparatus. This activity is accomplished by a calcium-dependent phosphorylation of aromatase, the key step in estradiol production. Phosphorylated aromatase increased estradiol production in a dose-dependent manner. Our data indicate that the ceramide cascade is essential for proper granulosa cell function and ceramide redistribution serves as a first step in order to proceed with the prosperous differentiation into a corpus luteum.

LOX-1 regulates estrogenesis via intracellular calcium release from bovine granulosa cells.

Estradiol produced by ovarian granulosa cells triggers the luteinizing hormone surge which in turn initiates ovulation in female mammals. Disturbances in estradiol production from granulosa cells are a major reason for reproductive dysfunctions in dairy cows. Endogenous estradiol production might be altered by reactive oxygen species (ROS) such as oxidized low-density lipoprotein (ox-LDL). Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a receptor of ox-LDL, leads to increased estrogenesis in granulosa cells. This activity is mediated by calcium release from endoplasmic reticulum (ER)-dependent and ER-independent calcium pools. Inhibition of the LOX-1 signal transduction pathway is followed by mitochondrial alterations. The membrane potential ΔΨ increases and the ROS production decreases in mitochondria after blocking LOX-1. Our data indicate that blocking the LOX-1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in metabolically highly active female mammals and, therefore, to defend against reproductive dysfunctions in humans and animals.

Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini.

Endoplasmic reticulum (ER) stress leads to accumulation of un- or misfolded proteins inside the ER and initiates the unfolded protein response (UPR). Several UPR components are physiologically involved in pancreatic development and are pathophysiologically activated during acute pancreatitis. However, the exact role of ER stress in exocrine pancreatic acini is mainly unclear. The present study examined the effects of tauroursodeoxycholic acid (TUDCA), a known ER chaperone, on acinar function and UPR components. Isolated rat pancreatic acini were stimulated by increasing concentrations of cholecystokinin (CCK-8) with or without preincubation of TUDCA. UPR components were analyzed, including chaperone binding protein (BiP), protein kinase-like ER kinase (PERK), X-box binding protein (XBP)-1, c-Jun NH(2)-terminal kinase (JNK), CCAAT/enhancer binding protein homologues protein (CHOP), caspase 3 activation, and apoptosis. In addition, TUDCA effects were measured on amylase secretion, calcium signaling, trypsin, and cathepsin B activation. TUDCA preincubation led to a significant increase in amylase secretion after CCK-8 stimulation, a 50% reduction of intracellular trypsin activation, and reduced cathepsin B activity, although the effects for cathepsin B were not statistical significant. Furthermore, TUDCA prevented the CCK-8-induced BiP upregulation, diminished PERK and JNK phosphorylation, and prohibited the expression of CHOP, caspase 3 activation and apoptosis. XBP-1 splicing was not altered. ER stress response mechanisms are activated in pancreatic inflammation. Chemical chaperones enhance enzyme secretion of pancreatic acini, reduce ER stress responses, and attenuate ER stress-associated apoptosis. These data hint new perspectives for an employment of chemical chaperones in the therapy of acute pancreatitis.

Upregulation of TNF receptor type 2 in human and experimental renal allograft rejection.

An important role of TNF interacting with TNFR2 has been shown in different models of ischemic, nephrotoxic and immune-mediated renal injury. To systematically evaluate the expression of TNFR2 in renal allograft rejection, we investigated human renal allograft biopsies and, in addition, established an experimental transplantation model in rats to verify the human data under standardized conditions. The expression of TNFR2 was analyzed in 96 human renal allograft biopsies with different disease entities. In a 6-day and a 28-day experimental protocol, TNFR2 was examined in kidney specimens and in the urine of control, uni-nephrectomized and transplanted rats +/- cyclosporine treatment (n = 114). In human biopsies and in rat allografts on day 6 with acute allograft rejection, significantly elevated expression of TNFR2 was observed in tubular epithelial cells, podocytes, B cells and monocytes/macrophages. The expression level was associated with renal function. The TNFR2 expression level at day 28 was significantly lower compared to day 6. TNFR2 is markedly upregulated both in human and experimental acute renal allograft rejection. Our data are robust and consistent between different species, suggesting a role for TNFR2 in the early course of rejection.

The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP.

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge-independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p-Emp47p hybrid protein was mislocalized to the cell surface in the alpha-COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.

Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast.

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.

Percutaneous transluminal angioplasty as first-line treatment of transplant renal artery stenosis.

BACKGROUND: Transplant renal artery stenosis (TRAS) is a frequent complication after renal transplantation, however long-term follow-up data after interventional treatment are rare. PATIENTS: In our transplant center 11 of 264 consecutive renal transplant recipients (4.17%) were diagnosed with TRAS. In addition, TRAS occurred in 2 renal transplant recipients that had been transplanted at other centers but who had their follow-up examinations in our center. Either a rise of the serum creatinine level and/or worsened systemic hypertension or routine examination with color Doppler sonography were indications for further diagnostic workup. METHODS: Direct angiography of the transplant renal artery was performed followed by percutaneous transluminal angioplasty (PTA) after the diagnosis of TRAS was confirmed in all of these patients. RESULTS: The immediate success rate for PTA was 92.3% (12/13). Only 1 patient with a severe kinking of the transplant renal artery had to undergo surgery to restore renal function. No complications occurred after the interventions. Thereafter the patients were monitored for a mean observation period of 33.15 months. Serum creatinine levels were significantly lower after the intervention, and estimated glomerular filtration rate (eGFR) increased accordingly. With regard to blood pressure there was only a trend for lower blood pressure levels and less antihypertensive use, whereas the dose of the prescribed drugs decreased significantly with time after interventional treatment of TRAS. In addition, a long-lasting rise of the hemoglobin levels could also be demonstrated. CONCLUSION: In summary, the beneficial effect of PTA of TRAS on renal function is long-lasting. Therefore, PTA, usually combined with stent placement, should be first-line treatment in TRAS in all patients. Surgical revascularization is only warranted, if PTA fails.

Severe renal vein stenosis of a kidney transplant with beneficial clinical course after successful percutaneous stenting.

A 51-year-old renal transplant recipient presented with marked renal function deterioration 13 months after renal transplantation. After exclusion of ureteral obstruction, transplant artery stenosis and acute rejection, the diagnosis of a severe renal vein stenosis was made by an MR scan. After angiographic confirmation of the stenosis, treatment was attempted with percutaneous stent angioplasty. The long-term clinical course was favorable, with marked improvement in renal function. Transplant renal vein stenosis is a rare, but potentially curable, cause of renal allograft functional deterioration.

TSF3, a global regulatory protein that silences transcription of yeast GAL genes, also mediates repression by alpha 2 repressor and is identical to SIN4.

TSF3 encodes one of six (TSF1 to TSF6) recently identified global negative regulators of transcription in Saccharomyces cerevisiae. Mutant tsf3 strains exhibit defects in transcriptional silencing of the GAL1 promoter, allow expression from upstream activation sequence-less promoters, and exhibit pleiotropic defects in cell growth and development. Here we show that TSF3 is involved in transcriptional silencing mediated by the alpha 2 repressor and demonstrate that specific systems of transcriptional silencing may depend on the more global role of TSF3. Cloning and sequencing of TSF3 allowed us to predict a 974-amino-acid gene product identical to SIN4, a negative regulator of transcription of the HO (homothallism) mating type switching endonuclease. TSF3 disruptions are not lethal but result in phenotypes similar to those of the originally isolated alleles. Our results, together with those of Y. W. Jiang and D. J. Stillman (Mol. Cell. Biol. 12:4503-4514, 1992), suggest that TSF3 (SIN4) affects the function of the basal transcription apparatus, and this effect in turn alters the manner in which the latter responds to upstream regulatory proteins.

Five-year follow-up after late conversion from calcineurin inhibitors to sirolimus in patients with chronic renal allograft dysfunction.

BACKGROUND: Chronic allograft nephropathy (CAN) is, among others, caused by nephrotoxic side effects of calcineurin inhibitors (CNI), which are to date still the mainstay of immunosuppressive therapy. Sirolimus (SIR), an immunosuppressive compound without effects on glomerular perfusion, has been used in CNI-sparing immunosuppressive protocols. We report the 5-year follow-up of a prospective, controlled conversion study from CNI to SIR in patients with moderately to severely impaired renal function. METHODS: Twelve renal transplant recipients with moderately to severely impaired renal function (estimated glomerular filtration rate of 17 to 35 mL/min according to the MDRD formula), enrolled in a prospective, controlled 1-year pilot study were followed for 5 years. RESULTS: Three renal grafts (25%) were lost during the 5-year follow-up. Graft loss was due to noncompliance in one patient and to CAN in the other two patients. These two patients returned to dialysis 43 and 59 months after conversion, corresponding to 86 and 75 months after transplantation, respectively. Six of nine patients had a stable or even better renal function compared to the baseline. The lipid profile increased initially, but then remained stable over time. CONCLUSION: Conversion of immunosuppressive therapy from CNI to SIR in patients with impaired renal function more than 1 year after transplantation is feasible and safe yielding improved renal function in the majority of patients, which was sustained at 5 years follow-up.

Visualization of receptor-mediated endocytosis in yeast.

We studied the ligand-induced endocytosis of the yeast alpha-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to alpha-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Delta. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to alpha-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes.

Increase in renal vascular resistance after intake of cyclosporin A and tacrolimus and reversal by nitroglycerin spray: a study in patients with stable renal allograft function.

PURPOSE: Acute effects of drug administration on renal arterial resistance index (RI) are still discussed controversially. In our study we investigated the immediate effects of cyclosporin A (CyA) and tacrolimus (FK-506) on renal arterial resistance indices in patients with stable graft function after renal transplantation. Additionally we studied the effects of nitroglycerin spray on resistance indices. METHODS: RI was measured by color Doppler sonography at baseline, at 1 and 2 hours after intake of medication and 30 minutes after administration of nitroglycerin spray which followed the 2-hour measurement. 34 renal transplant recipients were examined. 16 patients received CyA, 18 patients received FK-506. Whole blood levels of calcineurin inhibitors were taken at each time point. Arterial blood pressure and heart rate were measured to assess possible systemic hemodynamic effects. RESULTS: Mean RI values increased significantly in both groups 1 hour after calcineurin inhibitor intake and remained still significantly elevated after 2 hours. There was no significant increase of mean arterial blood pressure nor was there any correlation between whole blood levels of calcineurin inhibitors and mean RI. 30 minutes after administration of nitroglycerin spray, mean RI values decreased significantly to a level even below baseline. Mean arterial blood pressure also decreased after administration of nitroglycerin. CONCLUSION: Renal RI values are markedly influenced by a recent intake of calcineurin inhibitors and vasoactive substances such as nitrates. This demonstrates the necessity of keeping standardized conditions when using RI as a tool in followup investigations after renal transplantation.

Conversion from calcineurin inhibitors to sirolimus in patients with chronic renal allograft dysfunction.

BACKGROUND: Chronic renal transplant dysfunction in part may be due to the nephrotoxic effects of calcineurin inhibitors, which are still the mainstay of immunosuppressive therapy. Sirolimus, a new immunosuppressive compound devoid of significant nephrotoxicity, might therefore exhibit beneficial effects when used in renal transplant recipients with graft dysfunction. METHODS: Twelve renal transplant recipients included in this study had all been receiving calcineurin inhibitors for more than 12 months, and were free of rejection for more than 12 months. However, they demonstrated moderate renal dysfunction with serum creatinine values ranging from 1.8 to 4.0 mg/dL (164 to 351 micromol/L). After reaching a sirolimus level of 10 to 20 ng/mL, calcineurin inhibitor therapy was withheld. RESULTS: One month after initiation of sirolimus therapy, all patients were off calcineurin inhibitors. The average daily sirolimus dosage was 5.8+/-3.4 mg. No acute rejection episode and no graft failure was observed. No patient required hemodialysis or admission to the hospital. Calculated creatinine clearance increased from 63.4+/-9.9 to 69.2+/-9.7 mL/min (P=.0368) and serum bicarbonate increased from 20.8+/-3.17 to 22.5+/-3.7 meq/L (P=.001). Serum cholesterol increased from 180+/-26.5 to 239+/-28.8 mg/dL (4.65+/-0.69 to 6.18+/-0.74 mmol/L, P<.001), triglycerides increased from 155+/-53 to 289+/-123 mg/dL (1.75+/-0.6 to 3.26+/-1.39 mmol/L) and low-density lipoprotein cholesterol increased from 99+/-32 to 131+/-25.1 mg/dL (2.56+/-0.83 to 3.39+/-0.65 mmol/L, P=.01). Arterial blood pressure remained well controlled (126+/-15.6/74+/-8.9 vs 134+/-16.8/83+/-9.7). CONCLUSION: Conversion from calcineurin inhibitor therapy to sirolimus in patients more than 1 year after transplantation with impaired organ function is feasible, safe, and associated with a trend toward improved renal function.

Pretransplant calcium levels have no predictive value for delayed graft function, long-term graft function, cardiovascular events, or graft and patient survival in renal transplantation.

BACKGROUND: Disorders of calcium homeostasis are one of the most common problems in patients with end-stage renal disease (ESRD). Elevated calcium levels increase the incidence of cardiovascular mortality in ESRD patients, and appear to be a risk factor for the occurrence of delayed graft function (DGF) after kidney transplantation. Therefore, we investigated the impact of pretransplant serum calcium levels on outcomes after kidney transplantation: DGF, acute rejection, graft function, and survival, as well as the incidence of cardiovascular events. METHODS: We studied 285 patients (96.9% of all transplanted patients) who underwent their first transplantation between 1995 and 2004. Demographic data were extracted from hospital records or were documented during follow-up; serum samples were collected at the time of transplantation. RESULTS: In our cohort the incidence of DGF was 16.5% and 35.4% of acute rejection episodes (ARE). However, pretransplant calcium levels were not related to DGF or ARE in our patient cohort. Furthermore, there was no correlation between pretransplant serum calcium level with the incidence of cardiovascular events or mortality, as well as graft function or survival. CONCLUSION: In our study population pretransplant calcium levels showed no effect on DGF, ARE rate, the occurrence of cardiovascular events or death, renal graft function, or survival. Therefore, pretransplant calcium level is not a helpful marker for risk stratification at the time of transplantation.

Structural elements of bactopterin from Pseudomonas carboxydoflava carbon monoxide dehydrogenase.

Bactopterin is a novel pterin occurring in bacterial molybdoenzymes as the organic portion of the molybdenum cofactor. Its structure is investigated here. The compound contains a single pterin ring and carries a side chain at carbon atom 6 of the pterin nucleus as indicated by the formation of pterin-6-carboxylic acid upon alkaline permanganate oxidation. Studies with phosphate-cleaving enzymes revealed the presence of two monophosphoric acid monoesters. The affinity of reduced bactopterin for thiol-Sepharose points to the presence of thiol(s) in active bactopterin.

Dose-response relationship of complementary radiotherapy following four cycles of combination chemotherapy in intermediate-stage Hodgkin's disease.

PURPOSE: To determine the appropriate irradiation dose after four cycles of modern combination chemotherapy in nonbulky involved field (IF/BF) and noninvolved extended-field (EF/IF) sites in patients with intermediate-stage Hodgkin's disease (HD). MATERIALS AND METHODS: HD patients in stage I to IIIA with a large mediastinal mass, E stage, or massive spleen involvement were treated with two double cycles of alternating cyclophosphamide, vincristine, procarbazine, and prednisone (COPP) plus doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by EF irradiation in two successive trials (HD1 and HD5). In the HD1 trial (1983 to 1988), 146 patients who responded to chemotherapy were randomized to receive 20 Gy (70 patients) or 40 Gy (76 patients) of EF irradiation in all fields outside bulky disease sites. A cohort of 111 patients who fulfilled the same inclusion criteria in the subsequent trial HD5 (1988 to 1993) were treated with 30 Gy. Bulky disease always received 40 Gy. RESULTS: Freedom-from-treatment-failure (FFTF) and survival (SV) curves showed no differences between the 20-, 30-, and 40-Gy groups. However, acute toxicities were more frequent in the 40-Gy arm. Analysis of relapse patterns showed that 18 of 26 relapsing patients either failed to respond in initial bulky sites (n = 5) or had an extranodal relapse (n = 9) or both (n = 4). After 5 years, the cumulative risk for relapse in bulky sites is 10%, despite 40 Gy of radiation. CONCLUSION: Our results strongly suggest that there is no relevant radiotherapy dose effect in the range between 20 Gy and 40 Gy in IF/BF and EF/IF after 4 months of modern polychemotherapy in patients with intermediate-stage HD. Relapse patterns indicate that patients destined to relapse need more systemic, rather than local, treatment. Based on our data, we conclude that 20 Gy is sufficient in EF/IF of intermediate-stage HD following four cycles of modern polychemotherapy.

Polar phospholipids from bovine endogenously oxidized low density lipoprotein interfere with follicular thecal function.

The role of endogenously oxidized low density lipoprotein (oxLDL) in follicular steroidogenic regulation is unknown. Information may be important in order to elucidate ovulatory dysregulation in disordered lipid metabolism. To obtain specific data, we studied the effect of polar phospholipids (PL) isolated from oxLDL with different endogenous levels of lipohydroperoxides (LHP) on the thecal expression of mRNA encoding steroidogenic enzymes and cyclooxygenase 2 (COX-2), and on the thecal production of superoxide and progesterone. Large (preovulatory) bovine follicles were used and analyses of thecal fragments from single follicles were performed by radioimmunoassays, chemiluminescence assays and quantitative RT-PCR. Basal concentration of mRNA for several lipoprotein receptors exceeded by about 10-times the basal level of mRNA encoding steroidogenic enzymes, suggesting that preovulatory theca receptors may favour uptake of oxLDL. PL (5-11 pmol phosphorus/ml) decreased (up to 0.5-times the control) progesterone synthesis, production of superoxide and levels of P450 cholesterol side chain cleavage (P450 scc), 3beta-hydroxysteroid dehydrogenase and COX-2 mRNA. Abundance of COX-2 transcripts in thecal tissue incubated with forskolin depended on the progesterone/17beta-oestradiol ratio of the follicle fluid, i.e. the previous microenvironment in vivo. PL effects were mimicked by the platelet-activating factor (PAF). WEB 2086, a PAF receptor blocker, did not always abolish these responses, suggesting that the effects were not mediated solely by this receptor. PAF interfered dose-dependently with LH-induced responses, indicating interference with LH signalling. PL from mildly oxidized LDL (0.5 nmol/ml LHP) tended to exert greater effects than PL from oxLDL containing 1.5 nmol/ml LHP. In consideration of the known physiologic role of progesterone, COX-2 and possibly superoxide, these results provide evidence for a potential of PL from oxLDL to induce ovulatory dysregulation and suggest that the extent of the LDL oxidation seems to be important for interfering with thecal responses to the preovulatory LH surge.

Effect of genetic variation on therapy with angiotensin converting enzyme inhibitors or angiotensin receptor blockers in dialysis patients.

INTRODUCTION: The role of interaction of polymorphisms in the Renin-Angiotensin-System (RAS) with angiotensin converting enzyme (ACE) or angiotensin receptor (AGTR1) inhibitors (RAS inhibitors) is unknown, as is the role of such therapy in end stage renal disease (ESRD) patients. METHODS: We enrolled all 445 prevalent patients with diabetic nephropathy receiving maintenance hemodialysis in 30 centers in Southern Germany from August 1999 to January 2000 for prospective survival analysis until December 2003. Blood pressure and medication was recorded at inclusion. We determined survival specific for allelic variants of the ACE (insertion/deletion), Angiotensinogen (M235T) and AGTR1 (A1166C) genes. The effect of therapy with RAS inhibitors at study inclusion was determined for the allelic variants of each gene. The primary end point was all cause mortality (ACM). RESULTS: For all polymorphisms, and for therapy with RAS inhibitors there was no significant effect on survival in the complete collective (n = 445), though there was an insignificant trend for improved survival in patients on AGTR1 antagonists. Increased ACM risk was associated with treatment with RAS inhibitors only in patients homozygous for the wild type AGTR1 1166A allele (HR 1.65, p = 0.01). For all other polymorphisms, therapy with RAS inhibitors had no significant effect on ACM, irrespective of genotype. Similar results were obtained in patients with systolic ventricular dysfunction. CONCLUSION: Our data do not show a survival advantage for type 2 diabetes hemodialysis patients receiving RAS inhibiting therapy. In addition, our data indicate that allelic variation in RAS genes and pharmacogenetic interaction with RAS inhibition does not affect mortality risk in diabetic hemodialysis patients.