Regional differences in HLA antigen and haplotype frequency distributions in Germany and their relevance to the optimization of hematopoietic stem cell donor recruitment.

We analyzed regional differences in human leukocyte antigen (HLA)-A, -B, and -DR antigen and haplotype frequencies based on a sample of approximately 320,000 German donors in order to identify regions that are especially suited for ongoing stem cell donor recruitment. Geographic partitioning was carried out by postal code regions. Analysis of genetic distances suggests the existence of three regional clusters in South (regions 6-9), East (0-1), and Northwest (2-5) Germany. The southern cluster shows most favorable characteristics with respect to haplotypic and phenotypic diversity and the occurrence of rare HLA antigens. The opposite behavior is shown by regions 2-4 of the northwestern cluster. As a result of lower HLA diversity, completeness of a regional donor file in region 4 with 100,000 donors would be higher than that of a file in region 7 with 170,000 donors. This fact shows the relevance of regional HLA differences for practical donor registry planning. Results such as those presented in this work can be used to diminish the problem of decreasing marginal benefit of donor recruitment, as more than 13 million donors are registered worldwide today.

Estimating unbiased haplotype frequencies from stem cell donor samples typed at heterogeneous resolutions: a practical study based on over 1 million German donors.

The human leukocyte antigen (HLA) distribution in donor registry data is typically nonrandom as, mostly for economical reasons, typing additional loci or resolving ambiguities is selectively performed based on the previously known HLA type. Analyzing a sample of over 1 million German stem cell donors, we practically show the extent of the bias caused by the restriction of the input data for HLA haplotype frequency (HF) estimation to subsets selected according to their higher HLA typing resolution and, conversely, the correctness of estimates based on unselected data with a methodology suitable for heterogeneous resolution. We discuss algorithmic aspects of this approach and, also because of the sample size, provide some new insights into the distribution of HLA-DRB1 alleles in the German population and the application of HFs in unrelated donor search.

Aging of registered stem cell donors: implications for donor recruitment.

With more than 11 million registered stem cell donors worldwide and limited resources for health systems, it seems questionable if investments in ongoing donor recruitment are useful. Since there is evidence that transplant outcomes are better with younger donors, the age distribution of registered donors is highly relevant in this context. One might argue that the usefulness of a donor file decreases if there is no new donor recruitment not only as a result of loss of donors who reach the age limit for donation but also since those donors who remain in the file get older. We established a multivariate model to quantify this effect and to estimate the number (designated R) of donors who must be recruited annually to keep donor file usefulness constant. The model is applied to real data from DKMS German Bone Marrow Donor Center. R exceeds the number of donors who reach the age limit by factors up to 7.3. The model can serve as an easy-to-use tool for strategic donor registry planning. Our results suggest that analyses regarding optimal size of donor registries should also include the age distribution of registered donors.

Selective recruitment of stem cell donors with rare human leukocyte antigen phenotypes.

Many patients in need of a hematopoietic stem cell transplant do not find a fully matching donor although more than 11,000,000 potential donors are registered worldwide. Therefore, it is relevant to recruit donors who add diversity to the donor pool. We present the 'Roots' approach that includes the selection of already registered donors with rare HLA phenotypes and the recruitment of relatives of these donors. Two projects (Roots A and B) with different donor selection criteria were carried out. HLA phenotype frequency distributions of new donors differ significantly from the respective control groups: 2.7% of Roots A donors versus 1.1% of the control group have an HLA-AB phenotype that is unique in the DKMS file (P=0.001). Additionally, 39.5% of Roots B donors but only 18.3% of the control group have a unique HLA-ABDR phenotype (P<0.001). Similar results are found when phenotypes that are at most available 10 times in the DKMS donor file are analyzed. The results show that the Roots approach is generally suited to increasing the ratio of donors with rare HLA phenotypes in a donor file. Additional costs of Roots donor recruitment seem justified through the ratio of recruited donors with rare HLA phenotypes.

Common and well-documented HLA alleles over all of Europe and within European sub-regions: A catalogue from the European Federation for Immunogenetics.

BACKGROUND: A catalogue of common and well-documented (CWD) human leukocyte antigen (HLA), previously established by the American Society for Histocompatibility and Immunogenetics (ASHI), is widely used as indicator for typing ambiguities to be resolved in tissue transplantation or for checking the universality of any HLA allele in the world. However, European population samples, which are characterized by a substantial level of genetic variation, are underrepresented in the ASHI catalogue. Therefore, the Population Genetics Working Group of the European Federation for Immunogenetics (EFI) has facilitated data collection for an European CWD catalogue. MATERIALS AND METHODS: To this end, 2nd-field HLA-A, -B, -C,- DRB1,- DQA1,- DQB1 and -DPB1 data of 77 to 121 European population samples (21 571-3 966 984 individuals) from 3 large databases, HLA-net/Gene[VA], allelefrequencies.net and DKMS, were analysed. RESULTS: The total number of CWD alleles is similar in the EFI (N = 1048) and ASHI (N = 1031) catalogues, but the former counts less common (N = 236 vs 377) and more well-documented (N = 812 vs 654) alleles than the latter, possibly reflecting differences in sample numbers and sizes. Interestingly, approximately half of the CWD alleles reported by EFI were not reported by ASHI and vice-versa, underlining the distinct features of the two catalogues. Also, although 78 common alleles are widely distributed across Europe, some alleles are only common within specific sub-regions, showing regional variability. CONCLUSION: Although the definition of CWD alleles itself is affected by different parameters, calling for current updates of the list, the EFI CWD catalogue provides new insights into European population genetics and will be a very useful tool for tissue-typing laboratories in and beyond Europe.

Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method.

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts.

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinites for fructose diphosphate and Mg-2+ and were similarly activated by EDTA, dithiothreitol and cystamine. Mg-2+, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.

Inhibition of dextromethorphan metabolism by moclobemide.

This pilot study was conducted to evaluate the potential of the new antidepressant moclobemide to inhibit the cytochrome enzyme P4502D6 (CYP2D6) using the cough suppressant dextromethorphan as a substrate in four extensive metabolizers (EM) of debrisoquine. The subjects received seven oral doses of 20 mg dextromethorphan at 4-h intervals over 2 days (1 and 2) and subsequently moclobemide (300 mg b.i.d.) for 9 days. On days 10 and 11, they received seven doses of 20 mg dextromethorphan in addition to moclobemide. During monotreatment and combined treatment, blood was collected on days 2 and 11, respectively, for determination of dextromethorphan and its demethylated metabolites using automated high-performance liquid chromatography with column switching. Concurrent administration of moclobemide markedly reduced the O-demethylation of dextromethorphan, whereas the N-demethylation of dextrorphan to hydroxymorphinan was not affected. The findings indicate that moclobemide can affect the pharmacokinetics of drugs that are mainly metabolized by CYP2D6.

Comparison of moclobemide with selective serotonin reuptake inhibitors (SSRIs) on sexual function in depressed adults. The Australian and German Study Groups.

OBJECTIVE: To compare the emergent sexual effects of moclobemide and selective serotonin reuptake inhibitors (SSRIs) during acute and maintenance therapy in routine practice. METHOD: 268 patients were evaluated for sexual function at baseline, 6 weeks, 3 and 6 months of treatment using physician ratings and self-rating questionnaires. Patients received moclobemide, an reversible monoamine oxidase A inhibitor (RIMA), or a SSRI (fluoxetine, fluvoxamine, paroxetine, sertraline). RESULTS: Baseline values were similar in all groups. Incidences of impairments of sexual functioning with treatment, whether clinically relevant or not, were 24.3% with moclobemide and 61.5% with SSRIs (physician ratings), with no significant tolerance to these effects. There was a suggestion of differences between the SSRIs in their specific dysfunctions they cause. SSRIs (21.6% of patients) had about ten times the moclobemide rate (1.9%) of sexual dysfunction reported as adverse events. Antidepressant efficacy was comparable between treatments. CONCLUSION: In patients for whom sexual function is important or sexual dysfunction is present, moclobemide should be considered a first line antidepressant.

The role of cytochrome P450 2D6 in the metabolism of moclobemide.

The metabolic fate of moclobemide (Ro 11-1163), a new reversible and selective inhibitor of monoamine oxidase type A (MAO-A), has been assessed in a pilot study in 2 debrisoquine poor metabolizers (PM) and 4 extensive metabolizers (EM) after multiple oral dosings of moclobemide with and without co-medication of dextromethorphan. Absorption and disposition parameters were not different between PM and EM. Concurrent application of dextromethorphan, a selective substrate of CYP2D6, did not affect the pharmacokinetics of moclobemide. These results indicate that the cytochromal isoenzyme CYP2D6 does not play a major role in the metabolic degradation of moclobemide. Limited CYP2D6 activities because of a genetic defect or co-medications with CYP2D6 substrates should therefore not give rise to elevated moclobemide blood levels.

Particular aspects of adverse event assessment in post marketing surveillance.

The main feature of observational studies is the representation of naturalistic treatment conditions. In contrast to clinical trials, they allow the evaluation and quantification of adverse event profiles of drugs under "real life" conditions. The price for this unquestionable chance is the proneness to distorting factors, which may aggravate the interpretation of the study results. Analysis of observational study results therefore has to control for potentially influential factors and reconsider possible alternatives explaining observed associations. The most important distorting factors, which should be taken into account during analysis and interpretation are under-reporting, event selection, bias, confounding and misusage? Authors and readers of such study results should be aware of this possible sources of error, in order to derive optimal benefit from this study approach.

Pressure shift freezing as potential alternative for generation of decellularized scaffolds.

Background. Protocols using chemical reagents for scaffold decellularization can cause changes in the properties of the matrix, depending on the type of tissue and the chemical reagent. Technologies using physical techniques may be possible alternatives for the production grafts with potential superior matrix characteristics. Material and Methods. We tested four different technologies for scaffold decellularization. Group 1: high hydrostatic pressure (HHP), 1 GPa; Group 2: pressure shift freezing (PSF); Group 3: pulsed electric fields (PEF); Group 4: control group: detergent (SDS). The degree of decellularization was assessed by histological analysis and the measurement of residual DNA. Results. Tissue treated with PSF showed a decellularization with a penetration depth (PD) of 1.5 mm and residual DNA content of 24% ± 3%. HHD treatment caused a PD of 0.2 mm with a residual DNA content of 28% ± .4%. PD in PEF was 0.5 mm, and the residual DNA content was 49% ± 7%. In the SDS group, PD was found to be 5 mm, and the DNA content was determined at 5% ± 2%. Conclusion. PSF showed promising results as a possible technique for scaffold decellularization. The penetration depth of PSF has to be optimized, and the mechanical as well as the biological characteristics of decellularized grafts have to be evaluated.

An update to the HLA Nomenclature Guidelines of the World Marrow Donor Association, 2012.

For more than two decades, international cooperation and information technology have been playing key roles in the identification of suitable unrelated donors and cord blood units for hematopoietic SCT. To ensure consistent coding and interpretation of HLA data among the linked computer systems, the World Marrow Donor Association has standardized the extensions of the World Health Organization (WHO) Nomenclature for factors of the HLA system applied in practice. The first version of this report published in 2007 has become the reference for the technical validation of HLA information on donors and patients in the context of search and matching and is used by registries of volunteer unrelated hematopoietic stem cell donors and umbilical cord blood banks throughout the world. The present update became necessary after the major revision of the WHO HLA nomenclature in April 2010. It now covers issues arising when alleles are withdrawn or renamed because of the continuous updating of the WHO HLA nomenclature. In addition, formal validation and interpretation rules for the so-called 'multiple allele codes' have been added.

World Marrow Donor Association framework for the implementation of HLA matching programs in hematopoietic stem cell donor registries and cord blood banks.

A major goal of the World Marrow Donor Association (WMDA) is to foster international transplants of hematopoietic stem cells through the establishment of guidelines and recommendations in this field. In this tradition, this study defines a comprehensive framework for HLA matching programs, which use intricate algorithms to rapidly select potential donors for a patient from a database and to present these donors in a prioritized list. Starting with the comparison of single HLA markers of the donor and the patient possibly obtained using different testing methodologies at different resolutions, the more complex matching of loci and phenotypes is inductively built up. The consensus of this international collaborative group describes the state of the art in the field and points out many important design options compatible with the best practice. This should help existing registries to review and validate the most critical part of their IT systems and newly created donor registries around the world to tackle one of their real challenges.

Criteria for initiation and evaluation of minority donor programs and application to the example of donors of Turkish descent in Germany.

Minority donor programs aim to improve access to unrelated hematopoietic SCT for specific ethnic groups through directed donor recruitment. We have developed criteria for initiation and evaluation of such programs and applied them to the situation of donors of Turkish descent in Germany, as well as a program by DKMS German Bone Marrow Center that targets this group. Criteria for program initiation include the number of accessible minority donors, potential impact on the chances of finding matching donors, and general access to unrelated transplantation for patients of the targeted group. Success criteria comprise number and availability of recruited donors, the effect of these donors on the HLA phenotype distribution of a donor file, and the number of donations resulting from the program. More than 40 000 donors of Turkish descent have been recruited within the analyzed program to date. Recruited minority donors show more favorable demographic characteristics but lower availability rates than do German donors. Although HLA haplotype distributions of Turkish and German donors differ considerably, patients with common Turkish HLA phenotypes should benefit from the German donor pool even without a specific minority program. The analysis of donations from minority donors, however, shows specific benefits for patients with rare HLA phenotypes.

[Topographical anatomy of the female pelvis in ultrasound]. / Die sonomorphologische Topographie des weiblichen Beckens.

AIM: Achieving a high quality gynaecological ultrasound examination requires thorough knowledge of topographic anatomy. To date, there are no guidelines for a standardised course of the examination. The goal of the study was to define exact planes by means of cross-sectional anatomy and then to standardise the gynaecological ultrasound examination with the transabdominal, introital and transvaginal technique. METHOD: We developed a software tool based on IDL (Interactive Data Language) for the female data set of the Visible Human Project which generates free determinable planes in the volume. The organs of the female pelvis were divided into landmark- and target structures according to the ultrasonic visibility and the variability of the position, shape and structure. From this, a course for the gynaecological ultrasound examination was created and verified on 65 patients each with an inconspicuous ultrasound finding. In addition, the average duration of the examination was determined. RESULTS: The landmark structures could be demonstrated in all patients. Five planes were defined for each technique, and the course of the whole examination with 15 exact planes was described. The average duration of the examination was 4.5 minutes. CONCLUSION: As of now, the digitally reconstructed anatomical illustrations have achieved the best image resolution and quality regardless of the position of the plane in the examination volume. The standardised course of the gynaecological ultrasound examination can serve as a basis for the improvement of training quality and the evaluation of a general gynaecological ultrasound screening.

Quality-monitoring of psychotropic drug therapy in post-marketing surveillance. Results of a drug utilization observation (DUO) study on moclobemide.

Thanks to their non-intervening nature, post-marketing drug utilization observation (DUO) studies reflect both the properties of the substance under investigation and doctors' treatment decisions, thus-in contrast with clinical trials-allowing the interactions of these two factors to be investigated. A DUO study involving the administration of moclobemide to 9419 patients investigated the prescribing behavior of about two-thirds of all general practitioners and internists in Germany prescribing moclobemide during the study period. The results obtained suggest that, on the whole, moclobemide is being prescribed knowledgeably and correctly for the treatment of the full spectrum of depressive disorders. The management of these patients tended to adhere strictly to existing treatment recommendations while at the same time revealing a certain degree of latitude for treatment optimization. This applies particularly to the utilization of the recommended dosage range, especially in severely depressed states, and the consistent, syndrome-based combination treatment with hypnotics/sedatives or neuroleptics in the respective patient groups indicated. Potential interactions-though comparatively few are known for moclobemide-were taken into account in most cases. The safety profile closely matches the experience gained from clinical trials. Despite the large sample size involved there was no evidence of any hitherto unknown serious events. The experience gained during this survey suggests that the DUO study is a suitable instrument for monitoring the use of an antidepressant in the early post-registration period, enabling action to be taken at an early stage to correct any systematic deviations from the conditions of registration.

[Modification of sexual functions by antidepressants]. / Die Beeinflussung sexueller Funktionen durch Antidepressiva.

Sexual dysfunctions appear to be frequently occurring adverse events in treatment with antidepressants. Due to methodological reasons, a reliable estimation of the frequency of such events is currently not yet possible. There is evidence, that antidepressants could be differentiated with respect to their potency and specificity for disturbances of certain sexual subfunctions according to their pharmacological profile. With SSRIs in particular impaired functions of orgasm and ejaculation can be observed. No deteriorations are reported for buproprion and an improvement of sexual dysfunctions within the course of treatment for moclobemide. Viloxazine and trazodone appear to possess marked stimulating effects on libido and erectile functions. Generally the incidence of sexual adverse events is underestimated, although there is a pronounced impact on patient compliance. Taking into account this well documented side effect, sexual impairments should be monitored carefully within antidepressive treatment.

Laparoscopic therapy of an intact primary ovarian pregnancy with ovarian hyperstimulation syndrome: case report.

A case of an intact primary ovarian pregnancy with ultrasonographic demonstration of heart motion following ovarian stimulation is presented. After preoperative ultrasonographic confirmation of an extrauterine pregnancy, proof of the ovarian localization was achieved by intra-operative ultrasonographic visualization during a diagnostic laparoscopy on post-menstrual day 48. A moderate ovarian hyperstimulation syndrome with a concomitant increase in size, vulnerability and vascularity of the ovaries presented an additional challenge for the surgical approach. However, thanks to the early diagnosis of the ectopic pregnancy localization, a laparoscopic organ-preserving removal of the intact ovarian pregnancy was successfully performed. In this way, the fertility of the patient, who had previously undergone contralateral ovariectomy, was preserved. To our knowledge, this represents the first such treatment to be reported in the medical literature. Improvements in diagnosis and therapy of ovarian pregnancy are reviewed.