Thrombogenicity and long-term cytokine removal capability of a novel asymmetric triacetate membrane hemofilter

Hemofilters applied in continuous renal replacement therapies (CRRTs) for the treatment of acute kidney injury must meet high standards in biocompatibility and permeability for middle and large molecules over extended treatment times. In general, cellulose-based membranes exhibit good biocompatibility and low fouling, and thus appear to be beneficial for CRRT. In this in vitro study, we compared a novel asymmetric cellulose triacetate (ATA) membrane with three synthetic membranes [polysulfone (PS), polyethersulfone (PES), and polyethylenimine-treated acrylonitrile/sodium methallyl sulfonate copolymer (AN69 ST)] regarding thrombogenicity and cytokine removal. For thrombogenicity assessment, we analyzed the thrombin–antithrombin complex (TAT) generation in human whole blood during 5 h recirculation and filtration. Sieving coefficients of interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α) were determined using human plasma as test fluid. ATA and AN69 ST membrane permeability were determined also during long-term experiments (48.5 h). ATA exhibited the lowest TAT generation (6.3 µg/L at 5 h), while AN69 ST induced a pronounced concentration increase (152.1 µg/L) and filter clogging during 4 out of 5 experiments. ATA (IL-8: 1.053; IL-6: 1.079; IL-10: 0.898; TNF-α: 0.493) and PES (0.973; 0.846; 0.468; 0.303) had the highest sieving coefficients, while PS (0.697; 0.100; 0.014; 0.012) and AN69 ST (N/A; 0.717; 0; 0.063) exhibited lower permeability. Long-term experiments revealed stronger fouling of the AN69 ST compared to the ATA membrane. We observed the highest permeability for the tested cytokines, the lowest thrombogenicity, and the lowest fouling with the ATA membrane. In CRRT, these factors may lead to increased therapy efficacy and lower incidence of coagulation-associated events.     

Telomere dysfunction causes alveolar stem cell failure

Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.Mutations in telomerase and telomere genes cause abnormal telomere shortening. Clinically, this molecular abnormality manifests in a spectrum of telomere syndromes that recapitulate features of age-associated pathology (1). In highly proliferative compartments, such as the bone marrow, telomere dysfunction causes stem cell exhaustion, and hematopoietic stem cell transplantation can reverse this pathology (1). More commonly, short telomeres predispose to adult-onset disease in the lung, a tissue that has slow cell turnover (1). Idiopathic pulmonary fibrosis and emphysema are the most prevalent clinical manifestations of human telomere syndromes and account for more than 80% of presentations (1, 2). The alveolar structures are preferentially affected in these disorders, and their pathology is marked by inflammation and mesenchymal abnormalities (3, 4). Affected patients are also exquisitely sensitive to pulmonary-toxic drugs, which are fatal even when there is no detectable baseline lung disease (1, 5).The mechanisms by which telomere defects provoke lung disease are not understood, but a number of observations have pointed to lung-intrinsic factors and epithelial dysfunction as candidate events (6–10). For example, in telomerase-null mice, DNA damage preferentially accumulates in the air-exposed epithelium after environmentally induced injury, such as with cigarette smoke (7). The additive effect of environmental injury and telomere dysfunction has been suggested to contribute to the susceptibility to emphysema seen in these mice (7). Moreover, humans that carry mutations in the surfactant protein C gene, SFTPC, which is expressed exclusively in type 2 alveolar epithelial cells (AEC2s), develop lung disease phenotypes similar to those seen in telomerase mutation carriers (10–12). Pulmonary fibrosis and emphysema patients have also been noted to have abnormally short telomeres in AEC2s (6, 7, 13). These observations, along with AEC2s’ regenerative capacity (14–16), led us to hypothesize that telomere dysfunction is sufficient to provoke AEC2 failure and that this event drives lung disease pathogenesis.One hurdle to modeling the consequences of telomere dysfunction in a cell type-specific manner is that laboratory mice have very long telomeres (17). In the absence of telomerase, telomere dysfunction can be generated only after several generations of breeding, precluding cell type-specific studies (18). To overcome this limitation, we designed two experimental systems. First we examined the role of telomere shortening in purified AEC2s in a stem cell assay ex vivo. For an in vivo system, we generated a model in which telomere dysfunction can be induced by deleting telomeric repeat-binding factor 2 (Trf2) (19, 20) exclusively in adult AEC2s. Trf2 functions to suppress the DNA damage response, and its loss leads to telomere dysfunction by uncapping, thus allowing cell type-specific studies within a single generation (19, 20). The latter surrogate model allowed us to test the consequences of acquired DNA damage and telomere dysfunction in the adult lung. We show here, in both late-generation telomerase-null mice and in a conditional mutant model, that telomere dysfunction restricted to AEC2s impairs stem cell function by inducing senescence. This program recapitulates the inflammatory responses and susceptibility to injury that are hallmarks of telomere-mediated lung disease.     

The clinical value of neutrophil extracellular traps

Neutrophil extracellular traps (NETs) have recently been discovered as a central part of antimicrobial innate immunity. In the meanwhile, evidence accumulated that NETs are also generated upon non-infectious stimuli in various clinical settings. In acute or chronic inflammatory disorders aberrantly enhanced NET formation and/or decreased NET degradation seems to correlate with disease outcome. This review summarizes current knowledge about the relation of NETs in a broad spectrum of clinical settings. Specifically, we focus on the importance of NETs as a predictive marker in severely ill patients and further, we speculate about the potential pathophysiology of NETs.     

Improvement of multiple organ functions in hepatorenal syndrome during albumin dialysis with the molecular adsorbent recirculating system.

Recently, significant improvement of renal function and prolongation of survival were reported in hepatorenal syndrome (HRS) patients treated with the Molecular Adsorbent Recirculating System (MARS). As no impact on extrarenal organ function was documented, this trial looked into multiple organ function changes during MARS in HRS patients. Eight HRS patients (4 male, mean age 42.1 years, range 30-58, all United Network for Organ Sharing [UNOS] status 2A) were treated intermittendly 4-14 times (total 47, mean 5.9 +/- 3.4) between 4 and 8 h/single treatment. The following changes were observed pre- and posttreatment: bilirubin 466 +/- 146 to 284 +/- 134 micromol/L, creatinine 380 +/- 182 to 163 +/- 119 micromol/L, urea 26.4 +/- 10.3 to 12.9 +/- 4.9 mmol/L, plasma sodium 127.5 +/- 7.7 to 137.5 +/- 4.8 mmol/L (all p < 0.01). Mean arterial pressure (MAP) increased from 71.9 +/- 12.8 to 95.6 +/- 7.8 Torr (p < 0.001). Oliguria or anuria, present in all patients, was successfully reverted. Ascites, present in all patients, was not detectable after the treatment period. The hepatic encephalopathy grade decreased from 2.8 +/- 0.8 to 0.8 +/- 0.7 (p < 0.0001). Child-Index decreased from 13.25 +/- 1.3 to 9.4 +/- 1.8 (p < 0.001). The hospital survival rate was 62%. One man underwent successful liver transplantation 18 months after the treatment. We conclude that MARS can improve multiple organ functions in patients with HRS.     

In vivo perivascular implantation of encapsulated packaging cells for prolonged retroviral gene transfer.

Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirus-producing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ beta-galactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of approximately 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy.