Experimental rat model for therapeutic retinal pigment epithelium transplantation—unequivocal microscopic identification of human donor cells by in situ hybridisation of human-specific Alu sequences

Transplantation of retinal pigment epithelial (RPE) cells is discussed as a possible therapeutic approach for retinal degeneration. Xenogeneic transplantation of human RPE cells in animal models has been studied extensively. Various methods have been used to identify the graft cells, but these methods interfere with cell behaviour so that the monitored physiological post-transplantation course may be influenced. In the present study, we applied a method for an unequivocal identification of the graft cells without interfering cell metabolism or behaviour using in situ hybridisation (ISH) of human specific Alu sequences. Visualisation of the strong extended nuclear signal of Alu sequences was much easier than that of the small nuclear signals of donor-specific sex chromosome probes. With Alu probe, even single graft cells can be identified and their development can be observed in short-term and long-term studies. With this procedure, we could prove that donor cells were injected correctly into the subretinal space by a special injection technique that we developed previously. In combination with immunohistochemistry, donor cells could be clearly discriminated from macrophages, which contained phagocytosed donor cell fragments. Application of these ISH methods for species-specific identification was valuable for follow-up-studies of RPE transplantation.     

Human defensins

Antimicrobial peptides are small, cationic, amphiphilic peptides of 12–50 amino acids with microbicidal activity against both bacteria and fungi. The eukaryotic antimicrobial peptides may be divided into four distinct groups according to their structural features: cysteine-free -helices, extended cysteine-free -helices with a predominance of one or two amino acids, loop structures with one intramolecular disulfide bond, and -sheet structures which are stabilised by two or three intramolecular disulfide bonds. Mammalian defensins are part of the last-mentioned group. The mammalian defensins can be subdivided into three main classes according to their structural differences: the -defensins, -defensins and the recently described -defensins. Mammalian -defensins are predominantly found in neutrophils and in small intestinal Paneth cells, whereas mammalian -defensins have been isolated from both leukocytes and epithelial cells. Recently, two novel human -defensins, human beta-defensin-3 (HBD-3), and human beta-defensin-4 (HBD-4) have been discovered. Similar to HBD-1 and HBD-2, HBD-3 has microbicidal activity towards the Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and the yeasts Candida albicans and Malassezia furfur. In addition, HBD-3 kills Gram-positive bacteria such as Streptococcus pyogenes or Staphylococcus aureus, including multi-resistant S. aureus strains, and even vancomycin-resistant Enterococcus faecium. In contrast to HBD-1 and HBD-2, significant expression of HBD-3 has been demonstrated in non-epithelial tissues, such as leukocytes, heart and skeletal muscle. HBD-4 is expressed in certain epithelia and in neutrophils. Its bactericidal activity against P. aeruginosa is stronger than that of the other known -defensins. Here we present an overview of human antimicrobial peptides with some emphasis on their antifungal properties.J.J. Schneider and A. Unholzer contributed equally to this work     

Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens (n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

Nierenersatz bei diabetischer Nephropathie

Zusammenfassung Bei 180 Patienten mit fortgeschrittener Nephropathie auf dem Boden eines Diabetes mellitus Typ I und 214 Patienten mit Typ II Diabetes mellitus wurde der Einfluß verschiedener Verfahren der Nierenersatztherapie (intermittierende Haemodialyse/Haemofiltration, intermittierende Peritonealdialyse und kontinuierliche ambulante Peritonealdialyse CAPD) auf Morbidität und Letalität untersucht.Hypertonie und proliferative Retinopathie wurden bei Patienten mit Diabetes mellitus Typ I durch CAPD und Haemofiltration günstiger beeinflußt als durch Haemodialyse und intermittierende Peritonealdialyse. Bei Typ II Diabetikern wiesen die einzelnen Verfahren keine Unterschiede hinsichtlich der Beinflussung der Hypertonie auf.Die Morbidität, gemessen als stationäre Behandlungsbedürftigkeit (Tage/Jahr der Nierenersatztherapie) zeigte nur bei Diabetes mellitus Typ II eine signifikant kürzere Behandlungsdauer unter der Haemofiltration im Vergleich zur Haemodialyse. Erwartungsgemäß bedurften Typ II Diabetiker durchschnittlich einer längeren stationären Behandlung als Patienten mit Diabetes mellitus Typ I.Insbesondere Hypertonie und Hypotonie erforderten bei den Haemodialysepatienten unabhängig vom Typ des Diabetes mellitus eine längere Hospitalisierung als bei den Kranken unter der Haemofiltrationsbehandlung.Die Letalität war bei Typ I Diabetikern am geringsten unter der Haemofiltration und nach Nierentransplantation, bei Diabetes mellitus Typ II fanden sich keine Unterschiede zwischen den einzelnen Behandlungsverfahren. Unabhängig vom Typ des Diabetes mellitus war die Letalität bei Patienten mit diabetischer Nephropathie beträchtlich höher als bei Gleichaltrigen mit Niereninsuffizienz nichtdiabetischer Genese.Aufgrund der Ergebnisse empfiehlt es sich, zur Behandlung der diabetischen Nephropathie im Spätstadium bevorzugt solche Verfahren der Nierenersatztherapie anzuwenden, bei denen starke Blutdruckschwankungen vermieden werden können.     

Area under the viraemia curve versus absolute viral load: utility for predicting symptomatic cytomegalovirus infections in kidney transplant patients

A novel approach to predicting symptomatic cytomegalovirus (CMV) infections combines the level and the duration of viraemia in a single parameter. Sixty-four kidney transplant recipients were monitored by quantitative shell vial culture, pp65 antigenaemia, and polymerase chain reaction (PCR) of leucocytes. The area under the curve (AUC) of each parameter was determined from the onset of viraemia to the beginning of antiviral treatment. The AUC values were significantly higher in symptomatic than in asymptomatic patients. For antigenaemia and PCR, optimal AUC thresholds for predicting symptomatic CMV infections were determined. They were superior to standard cutoff levels of absolute viral load in sensitivity, specificity, and positive and negative predictive value. In 8 of the 23 patients who became symptomatic, impending clinical features were indicated earlier by the AUC thresholds than by standard viral load. In conclusion, the concept of the AUC should facilitate identification of patients at risk of symptomatic CMV infection.