Autosomal dominant polycystic kidney disease (ADPKD)—mechanisms of cyst formation and renal failure*

None of the hypotheses proposed so far to explain cyst formation in autosomal dominant polycystic kidney disease (ADPKD) is entirely satisfactory, e.g. the theory of tubular obstruction by intraluminal polyps or dilatation of nephron segments as a consequence of abnormal compliance of the basement membrane. Recent in vitro studies show that (i) synthesis of basement membrane material is abnormal and that (ii) the direction of transepithelial resorptive flux into a secretory mode is reversed as a consequence of faulty insertion of Na, K-ATP'ase into the luminal membrane. It remains unclear why cystic transformation of a few percent of nephrons should cause endstage renal failure. Our clinical and experimental studies do not provide evidence to support some hypotheses proposed in the past, i.e. that renal parenchyma is compressed by expanding cysts and that glomeruli are overperfused. Our histological studies show that progression to endstage renal failure is associated with (i) progressive arteriolar lesions (out of proportion to the vascular lesions seen in extrarenal vascular beds; and (ii) progressive interstitial fibrosis. It appears that fibroblasts in ADPKD are particularly sensitive to platelet derived growth factor (PDGF) which is secreted by epithelial cells of the cyst wall in a paracrine fashion. In contrast to previous opinion, which was presumably skewed by ascertainment bias, it appears that not all, and perhaps not even a majority, of ADPKD patients progress to endstage renal failure. Factors related to progression are gender, family history and hypertension. Both abnormal sodium excretion and inappropriate renin secretion play a role in the genesis of hypertension. Elevated blood pressure, albeit within the normotensive range, is demonstrable even in prepubertal children. The involvement of renin in renal vasoconstriction of normotensive ADPKD patients suggests a particular role of ACE inhibitors in the management of these patients.     

Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron

Ferric uptake regulation protein (Fur) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. The function of Fur is not limited to iron homeostasis. A wide variety of genes involved in various mechanisms such as oxidative and acid stresses are under Fur control. Flavohemoglobin (Hmp) is an NO-detoxifying enzyme induced by NO and nitrosothiol compounds. Fur recently was found to regulate hmp in Salmonella typhimurium, and in Escherichia coli, the iron-chelating agent 2,2'-dipyridyl induces hmp expression. We now establish direct inhibition of E. coli Fur activity by NO. By using chromosomal Fur-regulated lacZ reporter fusion in E. coli, Fur activity is switched off by NO at micromolar concentration. In vitro Fur DNA-binding activity, as measured by protection of restriction site in aerobactin promoter, is directly sensitive to NO. NO reacts with Fe(II) in purified FeFur protein to form a S = 12 low-spin FeFur-NO complex with a g = 2.03 EPR signal. Appearance of the same EPR signal in NO-treated cells links nitrosylation of the iron with Fur inhibition. The nitrosylated Fur protein is still a dimer and is stable in anaerobiosis but slowly decays in air. This inhibition probably arises from a conformational switch, leading to an inactive dimeric protein. These data establish a link between control of iron metabolism and the response to NO effects.     

Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Chickens and pigs are the most important livestock species worldwide. They are not only important sources of food, but also valuable models for evolutionary biology and biomedical science. Pigs share a high anatomical and physiological similarity with humans and are an important species for translational biomedical research, for example, in the areas of cancer, diabetes, neurodegenerative, and cardiovascular diseases (1–3). They also resemble the human pathophenotype more closely than rodents. For example, pig models for familial adenomatous polyposis (FAP) develop polyps in the large intestine as observed in human patients (4), whereas mouse FAP models develop them in the small intestine (5). In contrast to mammals, chickens are phylogenetically distant vertebrates from humans, but they were instrumental in the field of developmental biology due to the easy access to the embryonated egg. They are used for studying neurological and cardiovascular functions (6–8) and provided key findings in B cell development and graft versus host responses (9–11). Genetically modified livestock species also hold great promise for agriculture by offering new approaches for disease control, such as genome-edited pigs resistant to Porcine Reproductive and Respiratory Syndrome or Avian Leucosis Virus (ALV)-resistant chickens (12–15).Due to the lack of fully functional embryonic stem cells, genetic engineering in pigs and chickens has been a laborious, inefficient, and time-consuming procedure (16). The generation of pigs with precise germline modifications required gene targeting in somatic cells followed by somatic cell nuclear transfer. This also is not practical in chickens, where precise alteration of the genome only became possible with recent improvements in the cultivation and manipulation of germline-competent primordial germ cells (PGCs) (17–19). These modified PGCs can be injected into the blood vessel system of stage 13 to 15 (Hamburger−Hamilton [HH]) embryos to produce germline chimeras and, by further breeding, genetically modified chickens.With the advent of synthetic endonucleases such as CRISPR-Cas9 efficiency of targeted germline modification has improved in both species (20–23). It still requires the generation and breeding of new founder lines, which is time consuming in large animals. To circumvent the need for generating germline-modified animals, attempts have been made to carry out genome editing directly in specific organs or tissues (24–27). But this has been hampered by the need to deliver both Cas9 and the required guide RNA (gRNA) and by the limited cargo capacity of viral vectors. To bypass this drawback, Cas9 transgenic mice have been generated, requiring delivery of only the respective gRNAs (28).Here, we describe the generation of both Cas9 transgenic pigs and chickens that ubiquitously express Cas9 endonuclease and provide proof of its function in vitro and in vivo. These animals provide an innovative and efficient model for in vivo genome editing to assess gene function in health and disease.     

Human platelets attenuate Aspergillus species via granule-dependent mechanisms

Using laser scanning microscopy, we investigated whether platelets are capable of internalizing Aspergillus conidia and examined Aspergillus-platelet adherence. The influence of platelets on fungal growth was evaluated by assessing galactomannan (GM) release, hyphal elongation, and colony size. A secretion assay with [(3)H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (p<.05), hyphal elongation (p<.001), colony size, pigmentation, and 5-HT release ( p<.05). A lack of antifungal effects was observed with the microfilament inhibitor cytochalasin D. Platelets attenuate the virulence of Aspergillus species in vitro on the basis of granule-dependent effects.     

ADAM10 regulates endothelial permeability and T-Cell transmigration by proteolysis of vascular endothelial cadherin

Vascular endothelial (VE)-cadherin is the major adhesion molecule of endothelial adherens junctions. It plays an essential role in controlling endothelial permeability, vascular integrity, leukocyte transmigration, and angiogenesis. Elevated levels of soluble VE-cadherin are associated with diseases like coronary atherosclerosis. Previous data showed that the extracellular domain of VE-cadherin is released by an unknown metalloprotease activity during apoptosis. In this study, we used gain-of-function analyses, inhibitor studies, and RNA interference experiments to analyze the proteolytic release of VE-cadherin in human umbilical vein endothelial cells (HUVECs). We found that VE-cadherin is specifically cleaved by the disintegrin and metalloprotease ADAM10 in its ectodomain, releasing a soluble fragment and generating a carboxyl-terminal membrane-bound stub, which is a substrate for a subsequent gamma-secretase cleavage. This ADAM10-mediated proteolysis could be induced by Ca(2+) influx and staurosporine treatment, indicating that ADAM10-mediated VE-cadherin cleavage contributes to the dissolution of adherens junctions during endothelial cell activation and apoptosis, respectively. In contrast, protein kinase C activation or inhibition did not modulate VE-cadherin processing. Increased ADAM10 expression was functionally associated with an increase in endothelial permeability. Remarkably, our data indicate that ADAM10 activity also contributes to the thrombin-induced decrease of endothelial cell-cell adhesion. Moreover, knockdown of ADAM10 in HUVECs as well as in T cells by small interfering RNA impaired T-cell transmigration. Taken together, our data identify ADAM10 as a novel regulator of vascular permeability and demonstrate a hitherto unknown function of ADAM10 in the regulation of VE-cadherin-dependent endothelial cell functions and leukocyte transendothelial migration.     

Current treatment strategies of the German Hodgkin Study Group (GHSG)

Abstract:  Hodgkin's Lymphoma (HL) has developed to one of the best curable human cancers and overall about 80% of patients experience long-term disease free survival. Therefore, current treatment strategies aim at further improving treatment outcome, thereby trying to by minimize therapy-induced complications, such as infertility, cardiopulmonary toxicity, and secondary malignancies. Ongoing trials investigate a reduction of chemotherapy in terms of dose or cycles given, and the application of lower radiation doses and smaller radiation fields. For patients with a specific high-risk profile, new approaches with more intense drug combinations are currently being investigated. Moreover, the advent of effective salvage high-dose therapy for relapsed disease and a better understanding of prognostic factors have further improved the management of HL. Here, we summarize current strategies of the German Hodgkin Study Group (GHSG) in diagnostics and treatment of primary and relapsed HL, together with recent approaches for specific subgroups of HL patients.