Adipose tissue collection from a living kidney donor; an experimental model for the research of atherosclerosis.

Thanks to an increased number of living-donor kidney transplants the IKEM transplant program offers the possibility of obtaining adipose tissue for scientific purposes from patients with varying degrees of atherosclerosis. Surgery mainly addresses vascular complications of this disease. On the other hand, surgery may also be the reason for the development and acceleration of atherosclerosis - for instance, acceleration of atherosclerosis in the living kidney donor, particularly if, although meeting internationally recognized donation criteria, the donor actually suffers from metabolic syndrome. The effort to refine the examinations of living kidney donors in terms of eliminating the risk of developing atherosclerosis is a long-term project. The aims are to determine the risk factors for living kidney donors and to prevent long-term complications after donation. The paper gives a detailed description of the technique of adipose tissue collection from a living kidney donor and of the experimental model for the research of atherosclerosis.The project has the potential to increase the safety of living kidney donation and to enhance our present knowledge of atherosclerosis development mechanisms.

Aorto-iliac endarterectomy: Old-fashioned or re-newed method?

INTRODUCTION: Aorto-iliac occlusive disease is best treated with endovascular angioplasty/stenting or surgical bypass, depending on disease severity. Aorto-iliac endarterectomy was frequently used until the 1980s. However, it can still be performed in cases of previous failure or contraindication of standard methods. The aim was a retrospective evaluation of a single-center case series of aorto-iliac endarterectomy. METHODS: Seven patients at mean age 60±8 years (5768 years) were treated by aorto-iliac endarterectomy between 2013 and 2018. Rutherford categories of leg ischemia were 2 (moderate claudication) 3x, 3 (severe claudication) 2x, 4 (rest pain) and 5 (toe gangrene). The reasons for endarterectomy approach were: late in-stent iliac occlusion in an oncology patient, failure or complication of previous endovascular treatment of short iliac stenosis 2×, high infection risk of prosthesis use in long iliac-femoral occlusion, and short iliac occlusions 3x. Two patients after previous organ transplant were on immunosuppression. RESULTS: Technical success rate was 100%. There was no peri-operative (30 days) death or amputation. Mean follow-up was 17 months (1.1 month3.3 year). One patient required additional tibial bypass 1 month after endarterectomy to heal foot gangrene. One patient developed symptomatic re-stenosis which was treated with iliac stenting 8 months after procedure. All patients clinically improved and recovered from leg ischemia. Two patients died of tumor with preserved limb 1.1 month and 3.1 years after procedure, respectively. Five remaining patients are asymptomatic with patent revascularization to date. CONCLUSION: Aorto-iliac endarterectomy is a vital alternative technique for revascularization in selected patients when other methods seem inappropriate. Key words: endarterectomy - peripheral arterial disease - iliac artery - abdominal aorta.

MiRNA-149 as a Candidate for Facial Clefting and Neural Crest Cell Migration.

Nonsyndromic cleft lip with or without palate (nsCL/P) ranks among the most common human birth defects and has a multifactorial etiology. Human neural crest cells (hNCC) make a substantial contribution to the formation of facial bone and cartilage and are a key cell type in terms of nsCL/P etiology. Based on increasing evidence for the role of noncoding regulatory mechanisms in nsCL/P, we investigated the role of hNCC-expressed microRNAs (miRNA) in cleft development. First, we conducted a systematic analysis of miRNAs expressed in human-induced pluripotent stem cell-derived hNCC using Affymetrix microarrays on cell lines established from 4 unaffected donors. These analyses identified 152 candidate miRNAs. Based on the hypothesis that candidate miRNA loci harbor genetic variation associated with nsCL/P risk, the genomic locations of these candidates were cross-referenced with data from a previous genome-wide association study of nsCL/P. Associated variants were reanalyzed in independent nsCL/P study populations. Jointly, the results suggest that miR-149 is implicated in nsCL/P etiology. Second, functional follow-up included in vitro overexpression and inhibition of miR-149 in hNCC and subsequent analyses at the molecular and phenotypic level. Using 3'RNA-Seq, we identified 604 differentially expressed (DE) genes in hNCC overexpressing miR-149 compared with untreated cells. These included TLR4 and JUNB, which are established targets of miR-149, and NOG, BMP4, and PAX6, which are reported nsCL/P candidate genes. Pathway analyses revealed that DE genes were enriched in pathways including regulation of cartilage development and NCC differentiation. At the cellular level, distinct hNCC migration patterns were observed in response to miR-149 overexpression. Our data suggest that miR-149 is involved in the etiology of nsCL/P via its role in hNCC migration.

Coagulation and inflammation. Molecular insights and diagnostic implications.

Overwhelming evidence has linked inflammatory disorders to a hypercoagulable state. In fact, thromboembolic complications are among the leading causes of disability and death in many acute and chronic inflammatory diseases. Despite this clinical knowledge, coagulation and immunity were long regarded as separate entities. Recent studies have unveiled molecular underpinnings of the intimate interconnection between both systems. The studies have clearly shown that distinct pro-inflammatory stimuli also activate the clotting cascade and that coagulation in turn modulates inflammatory signaling pathways. In this review, we use evidence from sepsis and inflammatory bowel diseases as a paradigm for acute and chronic inflammatory states in general and rise hypotheses how a systematic molecular understanding of the "inflammation-coagulation" crosstalk may result in novel diagnostic and therapeutic strategies that target the inflammation-induced hypercoagulable state.

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma.

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.

[Haemostaseologic effects of fractionated snake venoms]. / Wirkungen von Schlangengiftfraktionen auf das Gerinnungssystem.

Comparative investigation concerning gelfiltration as well as haemostaseologic analysis of venoms and venom fractions of some snakes (elapidae and viperidae) have shown that in elapidae an inhibition of coagulation is dominant whilst in viperidae the stimulation of coagulation is of importance. Our investigations produce a basis to select substances for activation of coagulation and substances for inhibition of coagulation. Under pharmacological viewpoints the data may produce information to use snake fractions for anticoagulation or for procoagulant therapy in bleeding tendency.

The Role of Noncoding Genetic Variation in Isolated Orofacial Clefts.

In the past decade, medical genetic research has generated multiple discoveries, many of which were obtained via genome-wide association studies (GWASs). A major GWAS finding is that the majority of risk variants for complex traits map to noncoding regions. This has resulted in a paradigm shift in terms of the interpretation of human genomic sequence variation, with more attention now being paid to what was previously termed "junk DNA." Translation of genetic findings into biologically meaningful results requires 1) large-scale and cell-specific efforts to annotate non-protein-coding regions and 2) the integration of comprehensive genomic data sets. However, this represents an enormous challenge, particularly in the case of human traits that arise during embryonic development, such as orofacial clefts (OFCs). OFC is a multifactorial trait and ranks among the most common of all human congenital malformations. These 2 attributes apply in particular to its isolated forms (nonsyndromic OFC [nsOFC]). Although genetic studies (including GWASs) have yielded novel insights into the genetic architecture of nsOFC, few data are available concerning causality and affected biological pathways. Reasons for this deficiency include the complex genetic architecture at risk loci and the limited availability of functional data sets from human tissues that represent relevant embryonic sites and time points. The present review summarizes current knowledge of the role of noncoding regions in nsOFC etiology. We describe the identification of genetic risk factors for nsOFC and several of the approaches used to identify causal variants at these loci. These strategies include the use of biological and genetic information from public databases, the assessment of the full spectrum of genetic variability within 1 locus, and comprehensive in vitro and in vivo experiments. This review also highlights the role of the emerging research field "functional genomics" and its increasing contribution to our biological understanding of nsOFC.

Human adipose tissue accumulation is associated with pro-inflammatory changes in subcutaneous rather than visceral adipose tissue.

The importance of the involvement of adipose tissue macrophage subpopulations in obesity-related disorders is well known from different animal models, but human data are scarcer. Subcutaneous (n=44) and visceral (n=52) adipose tissues of healthy living kidney donors were obtained during living donor nephrectomy. Stromal vascular fractions were isolated and analysed by flow cytometry using CD14, CD16, CD36 and CD163 antibodies. Total macrophage numbers in subcutaneous adipose tissue increased (P=0.02) with body mass index (BMI), with a similar increase seen in the proportion of phagocytic CD14+CD16+CD36high macrophages (P<0.01). On the other hand, there was an inverse correlation between anti-inflammatory CD14+CD16-CD163+ macrophages (P<0.05) and BMI. These correlations disappeared after excluding obese subjects (BMI ⩾30 kg m-2) from the analysis. Interestingly, none of these subpopulations were significantly related to BMI in visceral adipose tissue. Obesity per se is associated with distinct, highly phagocytic macrophage accumulation in human subcutaneous adipose tissue.

The effect of ectopic fat on graft function after living kidney transplantation.

Renal transplantation is associated with a large number of risk factors that can have an influence on early renal graft function (ERGF). One of these factors could be the increasing number of obese kidney donors. The mechanisms of reduced ERGF in obese kidney donors are still poorly understood. To that end, we compared ERGF in recipients with body mass index (BMI), perivascular fat and plasma inflammation markers of live kidney donors. We hypothesized that the BMI of donors would negatively correlate with an average increase of glomerular filtration rate (GFR) and that it would also be associated with increased perivascular and plasma inflammation markers in the first seven days after transplantation. Between January 2013 and December 2014, some 58 living kidney transplantation pairs were included in the study. Donor and recipient demographic data, preoperative BMI, blood C-reactive protein (CRP) and adiponectin levels, perivascular adipose tissue (PAT) samples and recipient blood creatinine levels were analyzed. The median CRP of donors was 0.68 mg/l (max: 8.66 mg/l, min: 0.33 mg/l), the median of M1 macrophages (CD14+CD16+) in one gram of PAT was 5940 (max: 41 100, min: 248) and the median of adiponectin was 411 930 pg/ml (max: 14 217 000, min: 167 300) in plasma. We did not find any association between early renal graft function and the percentage of M1 macrophages in donor perirenal adipose tissue (p=0.83, r=0.03, n=58), adiponectin (p=0.65, r=0.06, n=58) or CRP (p=0.16, r=0.2, n=58) in plasma. The obesity level of donors, expressed as BMI, did not correlate with early renal graft function in the first seven days after transplantation. The associations between ERGF and plasma and perivascular fat inflammation markers were not significant. We confirmed a negative correlation between the BMI of recipients and an average increase of GFR in the first seven days after transplantation (p<0.02, r=-0.325, N=58). We confirmed a negative correlation of adiponectin plasma concentration to the BMI of donors.

Macrophage phenotypes in the adipose tissue of postmenopausal women.

Atherosclerosis pathology is the interplay between high intravascular LDL particle concentration and monocyte/macrophage presence within the sub-endothelial space of the artery. In this project, phenotypes of macrophages connected with subclinical inflammation in adipose tissue of living kidney donors were studied. Samples of subcutaneous adipose tissue of living kidney donors (n=36) were exposed to collagenase. Stromal vascular fraction (SVF) was eluted from the samples, then labeled with monoclonal antibodies (anti-CD14 and anti-calprotectin), conjugated with fluorochromes and analyzed by flow cytometry. The positive correlation between the number of total macrophages and calprotectin-positive macrophages with BMI in the subcutaneous adipose tissue of postmenopausal women was demonstrated (p<0.05; R=0.43 and p<0.01; R=0.60), whereas no positive correlation in premenopausal women and men was shown. In conclusion, we documented a significant effect of BMI increase on the presence of total macrophages in adipose tissue of postmenopausal women, in contrast to premenopausal women. This difference was much more pronounced when proinflammatory macrophages with membrane-bound calprotectin were analyzed.

[Characterization and economic impact of medical patients presenting at the emergency department of an university hospital]. / Internistische Patienten in einer universitären Notaufnahme: Charakterisierung und ökonomische Bedeutung für das Gesamtklinikum.

BACKGROUND: The number of patients in German emergency departments has been rising for years. This means additional need of staff and infrastructure for hospitals. METHODS: In this monocentric retrospective analysis the patient population of the central emergency department (ZNA) at the university hospital Frankfurt was investigated. Major symptoms, diagnoses with respect to diagnose-related groups and modes of admission to the emergency department have been analyzed. RESULTS: During 3 months, a total of 7376 patients presented to the ZNA. Analysis focused on 2186 patients referred to the department of internal medicine: most patients presented spontaneously (50.6%), 38.2% were admitted by ambulance services, only 9.7% were admitted by a primary physician. 44.9% of these patients were hospitalized, mainly with cardiological, pneumological and gastroenterological disorders. The predominant major symptoms were acute chest pain (15.4%), abdominal pain (7.1%) and syncope or collapse (6.1%). Patients hospitalized via ZNA contributed 31.9% of the total revenues of internal medicine departments. 31.7% of all hospitalized patients were admitted to the hospital by the ZNA. CONCLUSION: Emergency departments become more and more a regular part of ambulatory patients health care and contribute efficiently to the economic revenue of hospitals.

cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria.

The Hrp type III protein secretion system is essential for pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. Expression of the hrp gene cluster is controlled by HrpG, a two-component response regulator, and HrpX, an AraC-type transcriptional activator. Using the cDNA-AFLP technique, 30 hrpG-induced (hgi) and five hrpG-repressed (hgr) cDNA fragments were identified, defining a large hrpG-regulon in X. campestris pv. vesicatoria. Expression of most genes in the hrpG-regulon was dependent on hrpX. Seven cDNA fragments map to the known hrp gene cluster and flanking regions. All other genes appear to be scattered over the chromosome and endogenous plasmids. Sequence analysis identified genes encoding putative extracellular proteases, a putative transcriptional regulator and XopJ and XopB (Xanthomonas outer proteins), homologues of YopJ from Yersinia spp. and the avirulence protein AvrPphD of Pseudomonas syringae respectively. XopB is secreted by the Hrp type III secretion system. Analysis of deletion mutants in several hgi genes revealed a new virulence locus. This study demonstrates that cDNA-AFLP is a powerful tool to study prokaryotic transcriptomes and to identify genes contributing to Xanthomonas virulence and putative effector proteins.