[Suture-free amniotic membrane transplantation]. / Nahtlose Amnionmembrantransplantation.

Amniotic membrane transplantation has been used very successfully in ophthalmology since the 1940s for treatment of surface disorders. Over the course of the years the indications for use have been continuously extended. In contrast the operative technique is predominantly invasive, i.e. the amniotic membrane is surgically sutured onto the surface of the eye. In order to avoid surgical trauma the authors have developed a device which allows the amniotic membrane to be stretched in a ring and the amniotic ring can then be placed suture-free on the eye surface. The authors are hopeful that the biological principle of the amniotic membrane can be better utilized by uncomplicated repetitive application.

[Secondary malignancies in urinary diversions]. / Sekundärmalignome in Harnableitungen.

In contrast to ureterosigmoidostomy no reliable clinical data exist for tumor risk in different forms of urinary diversion using isolated intestinal segments.In 44 German urological departments, operation frequencies, indications, patient age, and operation dates of the different forms of urinary diversion, operated between 1970 and 2007, could be registered. The secondary tumors up to 2009 were registered as well and related to the numbers of the different forms of urinary diversions resulting in tumor prevalences.In 17,758 urinary diversions 32 secondary tumors occurred. The tumor risk in ureterosigmoidostomy (22-fold) and cystoplasty (13-fold) is significantly higher than in other continent forms of urinary diversion such as neobladders or pouches (p<0.0001). The difference between ureterosigmoidostomy and cystoplasty is not significant, nor is the difference between ileocecal pouches (0.14%) and ileal neobladders (0.05%) (p=0.46). The tumor risk in ileocecal (1.26%) and colonic neobladders (1.43%) is significantly higher (p=0.0001) than in ileal neobladders (0.5%). Of the 16 tumors that occurred following ureterosigmoidostomy, 16 (94%) developed directly at the ureterocolonic borderline in contrast to only 50% following urinary diversions via isolated intestinal segments.From postoperative year 5 regular endoscopic controls of ureterosigmoidostomies, cystoplasties, and orthotopic (ileo-)colonic neobladders are necessary. In ileocecal pouches, regular endoscopy is necessary at least in the presence of symptoms or should be performed routinely at greater intervals. Following neobladders or conduits, only urethroscopies for urethral recurrence are necessary.

Applicability of cord blood-derived unrestricted somatic stem cells in tissue engineering concepts.

OBJECTIVES: Cell-based tissue engineering concepts are becoming an important therapeutic alternative in the treatment of traumatic or chronic skeletal diseases. Here, we have evaluated cord blood-derived unrestricted somatic stem cells (USSCs) for use in bone and cartilage repair strategies. METHODS AND RESULTS: This type of somatic stem cell can be generated from cord blood with a current rate of 29% and we have documented excellent proliferation potential to high passage numbers. The cells have an initial population doubling time of 39 h, which slightly decreased with increasing passage number, but cells maintained their proliferation abilities up to passage 23. Cells clearly differentiated towards chondrogenic, adipogenic and osteogenic lineage as shown by reverse transcription-polymerase chain reaction as well as by histological, biochemical and immunohistochemical stains. Differentiation potential of USSCs was observed at passage 6, passage 15 and passage 21. In addition, USSCs showed increased secretion of vascular endothelial growth factor (VEGF) during osteogenic differentiation, as well as expression of key markers of angiogenesis such as vascular endothelial growth factor receptor-2 and platelet/endothelial cell adhesion molecule. CONCLUSIONS: USSCs when transplanted into a bone defect might support the repair process not only by pure remineralization but also by installation of angiogenic environment.

[In vivo and in vitro bone regeneration from cord blood derived mesenchymal stem cells]. / In-vitro- und In-vivo-Knochenregenerierung durch mesenchymale Stammzellen aus dem Nabelschnurblut.

BACKGROUND: Mesenchymal stem cells with an osteoblastic differentiating potency are investigated in regard of probable tissue engineering for further clinical application. The following report describes the use of cord blood derived stem cells as an alternative to other stem cell populations for bone regenerating tissue engineering. METHODS: To demonstrate the multipotency of cord blood derived mesenchymal stem cells, unrestringated somatic stem cells (USSC) were isolated from cord blood and underwent an osteo-, chondro- and adipoblastic in vitro stimulation. To evaluate the osteoinductive potency of a porcine collagen I/III cell carrier USSC were incubated on this matrix. To investigate the in vivo effects of human USSC an athymic rat model was developed. These cells were transplanted into a femoral defect. RESULTS: Cord blood derived mesenchymal stem cells (USSC) have an in vitro multipotency and show adipo-, chondro- and osteogenic differentiation. The porcine collagen I/III carrier promoted an osteoblastic differentiation. USSC survived after xenotransplantation in an athymic rat and differentiated into osteoblasts filling the bony defect zone. CONCLUSION: Human USSC are a mesenchymal multipotent stem cell population that shows osteoblastic differentiation onto a collagen I/III carrier in vitro as well as in an athymic rat in vivo.

Follow-up measurements of Nevirapine plasma levels over a prolonged period.

Over a period of more than four years of treatment, 177 Nevirapine plasma levels were taken from 27 patients. The values showed a high inter-patient variability and a lower intra-patient variability. Differences in body weight turned out to be the main reason for inter-patient variability. Treatment over a prolonged period did not result in any change in plasma concentrations. Adjusting dosage by means of therapeutic drug monitoring would appear to be a reasonable way of maximising patient benefit from treatment.

Second German consensus on immunogenetic donor search for allotransplantation of hematopoietic stem cells.

The present paper summarizes the results of the second German consensus meeting on immunogenetic donor search for allotransplantation of hematopoietic stem cells held in Essen in November 1999 under the auspices of the German Society for Immunogenetics (DGI) and the German Working Party for Blood and Marrow Transplantation (DAG-KBT). Immunogeneticists and transplant physicians from all over the country agreed to update the national standards for: (1) search strategy including the role of unrelated and extended family donor search after unsuccessful core family donor search, (2) histocompatibility loci to be typed, (3) histocompatibility typing techniques to be used (HLA serology vs DNA-based HLA typing, cellular tests, serum cross-match), and (4) acceptable HLA mismatches in the context of a defined underlying disease, donor type, and conditioning regimen.

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German caucasoid patients and their corresponding 821 potential unrelated stem cell transplants.

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.

Haematopoietic transplant potential of unrelated and related cord blood: the first six years of the EUROCORD/NETCORD Bank Germany.

To date, human umbilical cord blood (CB) has been employed successfully in well over 1000 allogeneic (unrelated and sibling) stem cell transplantations. Because of primary limitations in volume and cell numbers, over 90% of these transplantations were performed in children. Therefore requests for well standardised cord blood units of high quality are now increasing constantly. Examination and standardisation of unrelated and related cord blood stem cell preparations and banking as well as their biological characterisation was already initiated in Düsseldorf in 1992. Hitherto a total of 3236 CB samples with a mean volume of 89 +/- 25 ml, a mean total number of nucleated cells (NC) of 10 +/- 5 x 10(8) and a mean number of CFU-GM of 6 +/- 5 x 10(5) have also been validated by haematological, immunological and microbiological criteria. In addition to that, 97 directed CB donations of siblings with a clinical indication have been characterised and banked along the same lines. All CB units were collected from the umbilical cord vein immediately after vaginal full term delivery or caesarean section, then frozen and stored in liquid nitrogen. 1940 CB units were stored unseparated, the other 1296 were volume reduced using Hetastarch (HES) with a mean recovery of 85 +/- 13% of the nucleated cells, 86 +/- 12% and 84 +/- 13% for CFC and CD34+ cells, respectively. Only 5.0 ml of a CB sample is required for routine laboratory testing as there are HLA-class I typing, HLA-class II typing by sequence specific oligonucleotide probes (PCR-SOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony forming assays, and CD34+ status as well as certain viral infections such as CMV, Hepatitis B, C, HIV, Parvo B19 by PCR technology before releasing the CB unit for transplantation. For apparent viral infections, maternal sera obtained at birth were tested for HBsAg, anti-HBc, anti-HCV, -HAV-(IgG, IgM), -HIV-1-2, -EBV- (IgG, IgM), -HTLVI-II, -CMV (IgM, IgG), toxoplasmosis and syphilis. Within the last three years a total of 4860 preliminary searches and 680 extended unit reports were submitted to the CB bank Düsseldorf by fax or World Wide Web. So far 68 unrelated and 3 related CB units were delivered. From these 70 have been transplanted in 30 different transplant centres world-wide. Until now the evaluation of the first 53 unrelated CB-transplantations was performed together with the EUROCORD transplant registry. Three patients were excluded from the analysis, since they received an unrelated CB-transplant for non-engraftment after previous allotransplants. The median patient age of these 50 patients was 5.0 years (range 0.3-44), the median weight 18 kg (range 4-70 kg). The majority of the patients transplanted for malignancies (66%) suffered from ALL (n = 19), AML (n = 7), CML (n = 4) and lymphoma (n = 2) with two third (75%) in an intermediate (2nd CR) or advanced stage of disease (> 2nd CR); 13 patients had metabolic diseases and immunodeficiencies and three had aplastic anaemia. All CB samples as well as the patients' blood samples were typed in Düsseldorf for HLA-class I by serology confirmed by PCR-SSP and by high resolution DNA typing for HLA-DRB1 and HLA-DQB1 alleles. 96% of the 50 patients receiving unrelated CB were mismatched at one or more HLA-antigens. 41 of the 50 patients transplanted with unrelated CB from Düsseldorf were evaluable for engraftment with an overall engraftment rate of 83%. According to the defined criteria of EUROCORD, 9 of the 50 patients were not evaluable for engraftment, since they died before day 60. The present median follow-up time is 14 months (1.4-38). The Kaplan-Meier estimate of survival at one year is 42 percent. The three paediatric patients after sibling CB transplantations (ALL, amegakaryocytic thrombocytopenia and CML) are alive with a follow-up time of 350, 379 days and 531 days. (ABSTRACT TRUNCATED)

[Testicular metastasis of prostatic carcinoma 3 years after subcapsular orchiectomy. A case report]. / Hodenmetastase eines Prostatakarzinoms drei Jahre nach subkapsulärer Orchiektomie. Ein Fallbericht.

Metastatic cancer to the testis is a rare phenomenon of prostate carcinoma with only 80 cases reported in the literature. Most of these secondary testicular tumors were diagnosed on routine pathohistological examination of testicular tissue after plastic orchiectomy. In none of these cases metachronous development of these metastases has been described. For the first time we report on a 75-year old patient who developed a prostate carcinoma metastatis to the right testicle three years after undergoing subcapsular orchiectomy. This case shows that the urologist has to think about a metastatic cancer when he sees a testicular tumor also after plastic orchiectomy.

Rapid method for successful HLA class I and II typing from cadaveric blood for direct matching in cornea transplantation.

BACKGROUND: The aim of the study was to establish fast methods for postmortem HLA class I and II typing of cornea donors using cadaveric blood. METHODS: The commercially available reagents Lymphokwik MN and Dynabeads were evaluated here to provide an enriched living mononuclear cell (MNC) population and B-cell population for HLA class I and II typing of cadaveric blood by serology. Cadaveric blood was obtained 1-80 h post mortem. After isolation of living B-cells and B-cell-depleted living MNC's, cells were serologically typed by double-fluorescence cytotoxicity assay for HLA class I and II antigens. RESULTS: In 373 (81%) of 461 cadaveric blood samples HLA class I typing, and in 36 (62%) of 56 cadaveric blood samples HLA-class II typing, by serology was successful and accomplished within 5 h. Results from the serological HLA class I typing were confirmed by the results of HLA class I typing by RNA-based sequencing in seven cases. To improve the HLA class II typing, DNA typing using PCR with sequence-specific primers was performed in 148 samples and reverse hybridization of PCR-amplified DNA to immobilized HLA class II specific primers in 270 samples. These data were confirmed by DNA-based sequencing in five cases and by sequence-specific oligonucleotide hybridization in all cases. CONCLUSIONS: These results lead to the following typing strategy: HLA class I typing should be performed by serology. HLA class II typing should be performed by DNA technology because of its relative independence of the quality of the blood sample. The strategy we have developed is very successful and fast for tissue typing post mortem, thus expanding the time available for ideal HLA matching, increasing the number of available HLA-matched corneas and therefore reducing the number of graft rejections.

Fundamental Studies of Fiber-Guided Soft Tissue Cutting by Means of Pulsed Midinfrared Lasers and their Application in Ureterotomy.

Fiber-guided ablation of soft tissue with pulsed holmium and thulium lasers was investigated for intraluminal incisions. A bare fiber/tissue-contact application system with a nearly tangential irradiation geometry was first used in vitro on porcine ureter tissue. The efficiency and precision of the method was analyzed for different laser and application parameters. The ablation dynamics in water and tissue was investigated by fast flash photography. Uniform cuts could be achieved with 200- and 318-µm fibers using a free-running holmium laser with a pulse repetition rate of 10 Hz and an average power of up to 4 W. The depth of the cuts could be increased by using a thulium laser with the same laser parameters. By reducing the pulse duration by one order of magnitude, the quality of the incisions was made more irregular, the zone of thermomechanical damage increased, and the cuts became deeper owing to the growing influence of cavitation on shorter laser pulse durations. In a first clinical trial, 20 patients underwent holmium laser therapy to reopen ureteral strictures. Neither bleeding nor other adverse effects due to the laser treatment occurred, showing IR laser ureterotomy to be a suitable and promising minimally invasive technique. © 1998 Society of Photo-Optical Instrumentation Engineers.

[Measurement of expired alcohol concentrations with a new electrochemical sensor. A model investigation to determine interference with volatile anesthetics and clinical application]. / Messung der Atem-Alkoholkonzentration mit einem neuen elektrochemischen Sensor. Modelluntersuchung zur Querempfindlichkeit gegenüber volatilen Anästhetika und klinische Anwendung.

UNLABELLED: Absorption of irrigating fluid in transurethral prostatic resection (TURP) and percutaneous nephrolitholapaxy (PNL) into veins or delayed absorption due to fluid extravasation may result in a TURP syndrome. The measurement of end-tidal breath alcohol concentration (et AC) as a monitor of absorption of irrigating fluid labelled with 2% ethanol is limited by the disturbance of infrared sensors by volatile anaesthetics and nitrous oxide (N2O) (Fig. 2). An electrochemical sensor is acceptable for this method. The aim of the present study was the evaluation of breath alcohol measurements using an electrochemical sensor device (Alcomed 3010, Envitec). The stability of the sensor in the presence of volatile anaesthetics was examined using a lung model. In a clinical investigation, the device was then applied to spontaneously breathing or mechanically ventilated patients inhaling volatile anaesthetics during endoscopic urological surgery. METHOD: A two-chamber lung model filled with water for performing noninvasive measurements at the mouth of a patient has already been introduced by Brunner et al. (Fig. 1). With the addition of different amounts of ethanol to the temperature-controlled water, a constant ethanol concentration is achievable in the air above the water that is dependent on adjustments of the ventilator. Increasing concentrations of volatile anaesthetics (isoflurane, enflurane, halothane, and sevoflurane) were added to the fresh gas flow (2 l O2/3 l N2O) and etACs were measured using the manually triggered self-absorbent electrochemical sensor. First, regression equations were established between breath alcohol concentrations and increased volatile anaesthetic concentrations. Regression equations were then established between end-tidal anaesthetic gas concentrations and vaporizer adjustments in order to rule out an influence of ethanol on the anaesthetic gas monitor Ultima V (Datex). In the clinical investigation, 53 intubated and ventilated patients (33 undergoing PNL, 20 undergoing TURP) and 48 patients breathing spontaneously (32 with inhalation anaesthesia, 16 with spinal anaesthesia) were investigated. The etAC was measured with the Alcomed 3010 and compared with gas-chromatographically registered blood alcohol concentrations (BAC). The study had previously been approved by the Ethical Committee of the Medical University of Luebeck. Patients with liver disease and a history of toxic abuse were excluded. Only one value per patient (maximum BAC) was included in the statistics in order to avoid a cluster effect. RESULTS: The lung model experiments demonstrated that the measurement of etAC with an electrochemical sensor is free of interference by volatile anaesthetics (Table 1). The slope of the regression between the measured alcohol concentration and increased concentrations of anaesthetics did not differ significantly from baseline values. The measurement of end-tidal anaesthetic concentrations was not significantly different from vaporizer adjustments in the presence of increased alcohol concentrations (Table 2). During the clinical investigation, a regression between etAC and BAC was determined for both groups. For the group of patients breathing spontaneously, the correlation coefficient was 0.961 and the regression equation revealed etAC = 0.5677*BAC-0.1303 (Fig. 5). However, in the group of ventilated patients a biphasic course was shown that was dependent on BAC (Fig. 6). At BAC < 0.4%, a similar correlation (r = 0.856) to the spontaneously breathing group could be seen (regression equation: etAC = 0.617*BAC-0.020). Above 0.4% BAC there was no acceptable correlation (r = 0.444, regression equation: etAC = 0.202*BAC+0.104). CONCLUSIONS: The tested electrochemical sensor does not interfere with volatile anaesthetics and N2O as demonstrated by a lung model. There is a good correlation between etAC and BAC measurements in patients breathing spontaneously with special regard to the slope of the regression (s = 0.57).

[Ureterorenoscopy in children]. / Ureterorenoskopie bei Kindern.

Ureterolithiasis is rare in children. Although ureterorenoscopy is needed only in few cases, because of the small number of children who suffer from ureteral stones, the development of small-calibre semi-flexible instruments for the treatment of any ureteral stones can be seen as a useful extension of the range of therapeutic options. The established technique used in adults can also be applied in children. It is recommended that endoscopic stone treatment should be carried out in children only by operators who can draw on a wide experience of such procedures in adults. The small-calibre instruments must be used and extreme caution is still required. We report on our experience with ureterorenoscopy in 12 children.

Selection of unrelated bone marrow donors by PCR-SSP typing and subsequent nonradioactive sequence-based typing for HLA DRB1/3/4/5, DQB1, and DPB1 alleles.

HLA incompatibility between bone marrow recipients and unrelated donors is one of the main obstacles in bone marrow transplantation. HLA class I and generic class II DR and DQ typing is generally performed by serology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Here, the final selection of serologically pretyped unrelated bone marrow donors by confirmatory PCR-SSP (PCR-sequence-specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marrow donors and their corresponding recipients were analyzed for HLA-DRB identity by PCR-SSP analysis. After solid-phase single-strand separation, direct sequencing of the allele- or group-specific DRB amplified products was performed by applying fluorophorlabelled sequencing primers. Electrophoretically separated sequencing products were detected by means of an automated DNA sequencer. Group-specific amplification and sequencing of DQB1 alleles was carried out for all potential bone marrow donors and recipients, while only the final donor-recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strategy in combination with direct sequencing of PCR products allows matching of bone marrow transplant pairs with the highest degree of reliability for the assessment of HLA class II identity.