Allogeneic stem cell transplantation for patients with advanced rhabdomyosarcoma: a retrospective assessment.

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (allo-SCT) may provide donor cytotoxic T cell-/NK cell-mediated disease control in patients with rhabdomyosarcoma (RMS). However, little is known about the prevalence of graft-vs-RMS effects and only a few case experiences have been reported. METHODS: We evaluated allo-SCT outcomes of 30 European Group for Blood and Marrow Transplantation (EBMT)-registered patients with advanced RMS regarding toxicity, progression-free survival (PFS) and overall survival (OS) after allo-SCT. Twenty patients were conditioned with reduced intensity and ten with high-dose chemotherapy. Twenty-three patients were transplanted with HLA-matched and seven with HLA-mismatched grafts. Three patients additionally received donor lymphocyte infusions (DLIs). Median follow-up was 9 months. RESULTS: Three-year OS was 20% (s.e.±8%) with a median survival time of 12 months. Cumulative risk of progression was 67% (s.e.±10%) and 11% (s.e.±6%) for death of complications. Thirteen patients developed acute graft-vs-host disease (GvHD) and five developed chronic GvHD. Eighteen patients died of disease and four of complications. Eight patients survived in complete remission (CR) (median: 44 months). No patients with residual disease before allo-SCT were converted to CR. CONCLUSION: The use of allo-SCT in patients with advanced RMS is currently experimental. In a subset of patients, it may constitute a valuable approach for consolidating CR, but this needs to be validated in prospective trials.

Human leukocyte antigen distribution in German Caucasians with advanced Ewing's sarcoma.

BACKGROUND: Risk stratification criteria for patients with Ewing's sarcoma family of tumors (ESFT) are still limited. We hypothesized divergent human leukocyte antigen (HLA) patterns in ESFT patients and compared HLA-A, -B and -DR phenotype frequencies of patients with advanced ESFT with those of healthy controls. PATIENTS: HLA types of all German Caucasian patients with advanced ESFT and available HLA-A, -B and -DR data registered in the European Group for Blood and Marrow Transplantation, Paediatric Registry for Stem Cell Transplantation and the MetaEICESS data bases (study group, n=30) were retrospectively compared with HLA types of healthy German stem cell donors (control group, n=8 862 for single HLA frequencies and n=8 839 for allele combinations). Study group patients had been immuno-typed due to eligibility for allogeneic stem cell transplantation for high risk of treatment failure, and thus constituted a selected subgroup of ESFT patients. RESULTS: After Bonferroni correction for multiple testing (PC), phenotype frequencies of HLA-A24 remained significantly higher in the study group compared to controls (PC<0.05). Furthermore, several HLA combinations were significantly more frequent in the study group compared to controls (all PC<0.05). CONCLUSION: We report an increased incidence of circumscribed HLA patterns in German Caucasians with advanced ESFT. The possible clinical significance of this observation has to be re-assessed in prospective trials comprising larger ESFT patient numbers of all risk groups.

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells.

BACKGROUND: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8(+) T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. METHODS: Following repetitive CHM1(319) (VIMPCSWWV) and EZH2(666) (YMCSFLFNL) peptide-driven stimulations with HLA-A 0201(+) dendritic cells (DC), allo-restricted HLA-A 0201(-) CD8(+) T cells were stained with HLA-A 0201/peptide multimers, sorted and expanded by limiting dilution. RESULTS: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A 0201 and killed HLA-A 0201(+) ET lines expressing the antigen while HLA-A 0201(-) ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2(-/-)γ(C)(-/-) mice. Within this context, we identified the CHM1(319) peptide as a new candidate target antigen for ET immunotherapy. CONCLUSION: These results clearly identify the ET-derived antigens, EZH2(666) and CHM1(319), as suitable targets for protective allo-restricted human CD8(+) T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation.

No improvement of survival with reduced- versus high-intensity conditioning for allogeneic stem cell transplants in Ewing tumor patients.

BACKGROUND: Outcomes of Ewing tumor (ET) patients treated with allogeneic stem cell transplantation (allo-SCT) were compared regarding the use of reduced-intensity conditioning (RIC) and high-intensity conditioning (HIC) regimens as well as human leukocyte antigen (HLA)-matched and HLA-mismatched grafts. PATIENTS AND METHODS: We retrospectively analyzed data of 87 ET patients from the European Group for Blood and Marrow Transplantation, Pediatric Registry for Stem Cell Transplantations, Asia Pacific Blood and Marrow Transplantation and MetaEICESS registries treated with allo-SCT. Fifty patients received RIC (group A) and 37 patients received HIC (group B). Twenty-four patients received HLA-mismatched grafts and 63 received HLA-matched grafts. RESULTS: Median overall survival was 7.9 months [±1.24, 95% confidence interval (CI) 5.44-10.31] for group A and 4.4 months (±1.06, 95% CI 2.29-6.43) for group B patients (P = 1.3). Death of complications (DOC) occurred in 4 of 50 (0.08) and death of disease (DOD) in 33 of 50 (0.66) group A and in 16 of 37 (0.43) and 17 of 37 (0.46) group B patients, respectively. DOC incidence was decreased (P < 0.01) and DOD/relapse increased (P < 0.01) in group A compared with group B. HLA mismatch was not generally associated with graft-versus-Ewing tumor effect (GvETE). CONCLUSIONS: There was no improvement of survival with RIC compared with HIC due to increased DOD/relapse incidence after RIC despite less DOC incidence. This implicates general absence of a clinically relevant GvETE with current protocols.

No difference in survival after HLA mismatched versus HLA matched allogeneic stem cell transplantation in Ewing sarcoma patients with advanced disease.

Patients with advanced Ewing sarcoma (AES) carry a poor prognosis. Retrospectively, we analyzed 66 AES patients treated with allogeneic stem cell transplantation (allo-SCT) receiving HLA-mismatched (group A, n = 39) versus HLA-matched grafts (group B, n = 27). Median age at diagnosis was 13 years, and 15 years (range 3-49 years) at allo-SCT. The two groups did not differ statistically in distribution of gender, age, remission status/number of relapses at allo-SCT, or risk stratum. 9/39 (23%) group A versus 2/27 (7%) group B patients developed severe acute graft versus host disease (GvHD). Of patients alive at day 100, 7/34 (21%) group A versus 9/19 (47%) group B patients had developed chronic GvHD. In group A, 33/39 (85%) versus 20/27 (74%) group B patients died of disease and 1/39 (3%) versus 1/27 (4%) patients died of complications, respectively. Altogether 12/66 (18%) patients survived in CR. Median EFS 24 months after allo-SCT was 20% in both groups, median OS was 27% (group A) versus 17% (group B), respectively. There was no difference in EFS and OS in AES patients transplanted with HLA-mismatched versus HLA-matched graft in univariate and multivariate analyses. In this analysis, CR at allo-SCT is a condition for survival (p < 0.02).

Transthyretin and complex protein pattern in aqueous humor of patients with primary open-angle glaucoma.

PURPOSE: To analyze protein patterns in the aqueous humor of glaucoma patients in comparison to control subject using two different methods. METHODS: Aqueous humor was collected from 52 patients with primary open-angle glaucoma (POAG) and from 55 control subjects (CO). Twenty-two POAG samples and 24 CO samples were used for protein profiling through surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) ProteinChip arrays. The data were analyzed by multivariate statistical methods and artificial neural networks. One highly significant biomarker was identified through matrix assisted laser desorption/ionisation time of flight-mass spectrometry (MALDI-TOF). Thirty samples from patients with POAG and 31 control samples were analyzed through two-dimensional electrophoresis. Subsequently, the protein spots of all gels were detected, and the two groups were compared. One spot group exhibiting clear differential abundance was identified by mass spectrometry (electrospray ionization mass spectrometry). RESULTS: In the samples analyzed by SELDI-TOF-MS, about 250 protein peaks could be consistently clustered in both groups. The analyses revealed eight biomarkers, which discriminated glaucoma from non-glaucoma controls with a sensitivity of 90% and a specificity of 87%. These biomarkers were purified further, and one marker, which was upregulated in glaucoma patients (p=0.006), was identified as transthyretin. The upregulation of transthyretin in POAG patients was also confirmed by enzyme linked immunosorbent assay (ELISA; p=0.03). In all samples analyzed by two-dimensional electrophoresis, complex protein patterns were detected in a total of 177 spot groups. The aqueous humor of all glaucoma patients revealed some regions that were clearly different from the controls. Several spots were significantly increased in the aqueous humor of glaucoma patients. One of the proteins that is highly abundant in the aqueous of glaucoma patients was identified as transthyretin. CONCLUSIONS: The aqueous humor of glaucoma patients revealed characteristic differences in protein/peptide profiles from control patients using two different analytical methods, SELDI-TOF-MS and two-dimensional electrophoresis. Interestingly, we could detect elevated transthyretin concentrations in glaucoma samples. Transthyretin might play a role in the onset of glaucoma since it has been shown to form amyloid deposits. These particles could cause outflow obstructions thereby increasing intraocular pressure as a possible onset mechanism.

Membrane-bound proteindisulfide isomerase (PDI) is involved in regulation of surface expression of thiols and drug sensitivity of B-CLL cells.

The proteindisulfide isomerase (PDI), a multifunctional cytoplasmic enzyme with additional chaperone activity, has been shown recently, using monoclonal antibodies, to be located on the membrane of mature human B lymphocytes and B cell chronic lymphocytic leukemia (B-CLL) cells. Here, evidence is presented that this antigen exhibits catalytic activity as measured by the reductive degradation of insulin (release of A chain molecules) on intact B cells in patients suffering from B-CLL, as well as on JVM 13 cells (B-CLL cell line). More than 98% of these cells exhibited PDI activity which could be inhibited by bacitracin and also by monoclonal and polyclonal antibodies to PDI. Interestingly, surface PDI expression was strongly correlated in our study with protein-bound membrane SH groups. These surface protein thiols were specifically determined by using low concentrations of the chloromethyl-derivative based fluorescent probe 5-(and6)-(((4-chloromethyl)-benzoyl)amino)-tetramethyl-rhodamine (CMTMR) at low temperature in the presence of sodium azide in flow cytometry. The highest PDI and SH expression was found on B lymphocytes, particularly B-CLL cells. The mean fluorescence intensity (MFI) of CMTMR-positive B cells in the B-CLL line was up to 10-fold higher than that of controls, indicating a strong elevation of cell membrane-located protein thiols on malignant B cells. The link between PDI and SH expression on cell surfaces points to a functional interaction between the two. Treatment with bacitracin resulted in a strong inhibition of PDI and a dramatic increase in surface protein thiol expression of B-CLL cells. Similar effects could be observed by cell treatment with anti-PDI antibodies, indicating that this enzyme system plays a crucial role in the regulation of protein-bound SH groups. Interestingly, artificially induced protein thiol expression led to significantly higher cellular resistance to the cytostatic drugs chlorambucil, vinblastin, and cisplatin in vitro as measured by cell growth. These data suggest for the first time a regulatory effect of PDI on the surface protein thiol status of B cells. The increased expression of PDI may play a crucial role in SH-mediated protection and drug resistance in malignant B lymphocytes.

Evidence of a defective thiol status of alveolar macrophages from COPD patients and smokers. Chronic obstructive pulmonary disease.

In increasing numbers of pulmonary diseases an association with a loss of intracellular thiols, mainly glutathione, is postulated. Therefore, the quantitative measurement of thiols within different viable cells is a possible metabolic parameter for cellular function and defense capacity of all pulmonary immune cells including alveolar macrophages (AM), that are highly compromised by oxidative stress. In this study the cellular thiol content was determined using fluorochrom conjugated chloromethyl derivatives (5-chloromethylfluorescein diacetate, CMFDA) in flow cytometry. The procedure was evaluated in vitro using biochemical techniques for glutathione quantification. Based on this approach, AM obtained from bronchoalveolar lavage (BAL) of smokers and patients with chronic obstructive pulmonary disease (COPD) showed a significant thiol deficiency compared to a nonsmoker/non-COPD group. The cellular thiol expression of AM from smokers and COPD patients reached only 50 and 53% of the control group. Lowest thiol concentrations (47% of control) were detected within the smoker(+)/COPD(+) group. This intracellular thiol deficiency significantly correlated with reduced lung function (FEV(1), PaO(2)). With regard to the tightly regulated thiol metabolism of immune cells, these results imply the onset of functional disturbances in thiol deficient AM. The determination of the cellular thiol content of AM, obtained from BAL by flow cytometry, presents a simple and reliable tool to monitor the effect of therapeutic measures focusing on the stabilization of the cellular thiol status.

Restoration of the cellular thiol status of peritoneal macrophages from CAPD patients by the flavonoids silibinin and silymarin.

During continuous ambulatory peritoneal dialysis (CAPD) the peritoneal immune cells, mainly macrophages, are highly compromised by multiple factors including oxidative stress, resulting in a loss of functional activity. One reason for the increase of inflammatory reactions could be an imbalance in the thiol-disulfide status. Here, the possible protective effects of the antioxidant flavonoid complex silymarin and its major component silibinin on the cellular thiol status were investigated. Peritoneal macrophages from dialysis fluid of 30 CAPD patients were treated with silymarin or silibinin up to 35 days. A time-dependent increase of intracellular thiols was observed with a nearly linear increment up to 2.5-fold after 96 hours, reaching a maximum of 3.5-fold after 20 days of culture. Surface-located thiols were also elevated. The stabilization of the cellular thiol status was followed by an improvement of phagocytosis and the degree of maturation as well as significant changes in the synthesis of IL-6 and IL-1ra. Furthermore, the treatment of peritoneal macrophages with flavonoids in combination with cysteine donors resulted in a shortened and more efficient time course of thiol normalization as well as in a further increased phagocytosis. In addition, GSH-depletion in thiol-deficient media simulating CAPD procedures led to intracellular thiol deficiency similar to the in vivo situation. It is concluded that treatment with milk thistle extracts silymarin and silibinin alone or, more effectively in combination with cysteine donors, provide a benefit for peritoneal macrophages of CAPD-patients due to a normalization and activation of the cellular thiol status followed by a restoration of specific functional capabilities.

Induction of protein disulphide-isomerase and immunoglobulins by pokeweed mitogen in human lymphocytes.

The Protein disulphide-isomerase (PDI, EC 5.3.4.1, Thiol-proteindisulphide oxidoreductase, EC 1.8.4.2) is thought to regulate the sulfhydryl status of cells and to catalyze thiol/disulphide exchange reactions involved in the post-translational processing of disulphide containing secretory proteins. The aim of the present investigations was to study the possible function of this enzyme in differentiation of B lymphocytes and immunoglobulin synthesis. Non-adherent human mononuclear cells or purified T cells were cultured in presence and absence of Pokeweed mitogen over 3, 5 and 7 days. Monoclonal antibodies and a rabbit polyclonal antiserum specific for human liver PDI were produced to determine the concentration of PDI by an ELISA technique and cytoplasmic immunofluorescence. After PWM stimulation, both, the cellular content of PDI as well as that of immunoglobulin, particularly IgM, have been found to be induced in a time dependent manner with a 2-3 fold increase in comparison to unstimulated cells. The specific induction of PDI in human B lymphocytes was also confirmed in Western blotting. Our findings suggest that PDI plays a critical role in the final stages of B cell differentiation and immunoglobulin synthesis by activated B cells and plasma cells, respectively.

[European Association of Urology guidelines on urinary and male genital tract infections]. / Synoptische Leitlinie der sexuell übertragbaren Erkrankungen (STD) mit Primärsymptomen im männlichen Genitale (S1). Leitinien der Deutschen Gesellschaft für Urologie.

Today, the classical bacteria that cause venereal diseases, e.g. gonorrhea, syphilis, chancroid and inguinal granuloma, only account for a small proportion of all known sexually transmitted diseases (STDs). Other bacteria and viruses as well as yeasts, protozoa and epizoa must also be regarded as causative organisms of STD. Taken together, all sexually transmitted infections comprise more than 30 relevant STD pathogens. However, not all pathogens that can be sexually transmitted manifest diseases in the genitals and not all infections of the genitals are exclusively sexually transmitted. Concise information and tables summarising the diagnostic and therapeutic management of STDs in the field of urology allow a synoptic overview, and are in agreement with the recent international guidelines of other specialist areas. Special considerations (i.e. HIV infection, pregnancy, infants, allergy) and recommended regimens are presented.

Total body MRI-governed involved compartment irradiation combined with high-dose chemotherapy and stem cell rescue improves long-term survival in Ewing tumor patients with multiple primary bone metastases.

We examined the role of total body magnetic resonance imaging (TB-MRI)-governed involved compartment irradiation (ICI) and high-dose chemotherapy (HDC), followed by stem cell rescue (SCR) in patients with high-risk Ewing tumors (ETs) with multiple primary bone metastases (high-risk ET-MBM). Eleven patients with high-risk ET-MBM receiving initial assessment of involved bones by TB-MRI were registered from 1995 to 2000 (group A). In all, 6 patients out of 11 had additional lung disease at initial diagnosis; all had multifocal bone disease with more than three bones involved. After systemic induction with etoposide, vincristine, adriamycin (doxorubicin), ifosfamide, and actinomycin D (EVAIA) or VAIA chemotherapy, ICI of all sites positive by TB-MRI was administered, followed by HDC and SCR. A second group matched for observation period and consisting of 26 patients with more than three involved bones at diagnosis was treated with the European Intergroup Cooperative Ewing Sarcoma Study-92 (EICESS-92) protocol (group B). These patients did not receive TB-MRI and consequently did not receive TB-MRI-governed ICI, or HDC and SCR. Survival in group A vs group B was 45 vs 8% at 5 years and 27 vs 8% at 10 years after diagnosis (log rank and Breslow: P<0.005). We conclude that TB-MRI-governed ICI followed by HDC and SCR in ET-MBM is feasible and warrants further evaluation in prospective studies.

[Results of partial kidney resection in nephrolithiasis]. / Ergebnisse nach Nierenteilresektionen bei Nephrolithiasis.

In order to establish the actual recidivation rate, the patients with partial resections of the kidneys of the years 1965 to 1978 were examined. Of 120 patients treated 106 could be examined for the second time, at a mean time of secondary examination of 6.5 years. 24.5% of the patients had a recidivation at the time of the examination. The annual recidivation rate was calculated depending on the examination. It increases with the duration of the time of the after-examination. The comparison with the recidivation number after pyelolithotomy (26.7%) does not show an evident effect of the partial resection of the kidney on the frequency of recidivations. In the judgment, however, the selection of these patients for this operation technique is to be taken into consideration. The use of the coagulum pyelolithotomy has led to a more stringent indication for the partial resection of the kidney.

[Therapy of bladder calculi--trends and results of a mainly transurethral oriented clinic]. / Die Therapie der Blasensteine--Trends und Ergebnisse einer vorwiegend transurethral orientierten Klinik.

From 1975 to 1985 519 patients with urinary calculi were treated in the Urologic Clinic of the Berlin-Buch Hospitals. 57.6% of them were treated by lithotripsy and TUR-P, 14.8% by lithotripsy alone, 11.8% by stone removal and transvesical adenomectomy and 15.8% by suprapubic cystotomy only. The diagnosis of the four forms of therapy is described. The trend in the conception of treatment goes to the treatment of calculi transurethrally in combination with the simultaneous removal of the obstruction of the outflow from the urinary bladder. Since October 1985 the electrohydraulic lithotripsy has been used. Up to 1985 in 218 patients urinary calculi were crushed by means of the device Urat-1. In 190 patients (87.3%) the lithotripsy was carried out in combination with other therapeutic measures, in 153 (70.2%) a transurethral resection of the prostate gland and in 28 patients (12.9%) other transurethral interventions were performed. Postoperatively, in 7 patients complications occurred (3.2%), perforations of the urinary bladder were not seen. Taking into consideration the whole number of patients, it is reported on the advantages of the use of the electrohydraulic lithotripsy by means of the apparatus Urat-1 as an uncomplicated technique easily to be learned, which excellently may be combined with simultaneous transurethral interventions.

[Transurethral electro-resection in bladder cancer. Indications and results]. / Die transurethrale Elektroresektion beim Blasenkarzinom. Indikationsstellung und Ergebnisse.

On principle every vesical tumour should be resected at first transurethrally. The TUTUR allows an exact establishment of the penetration depth of the tumour. The transurethral electro-resection gives representative tissue for the histological examination. For the majority of the superficially infiltrating tumours it represents the method of choice with curative aim. In the treatment of the progressed vesical carcinomas the TUTUR with palliative aim gives immense advantages. Using the developing paravesical inflammation wall the following secondary (and more) resection into the perivesical fatty tissue is possible. The intervention can be repeated at every time it is not mutilating does not cause any stoma problems or metabolic derangements as after discharge of the urine and may be suggested to patients in a general condition which forbids an open surgical intervention. The resection can improve the quality of the patient's life for a long time without great stress and thorough changes of the life habits of old people. However the resection treatment will be successful only then, when it will be performed by an experienced (in transurethral operations) operator with a technically good instrumentarium.

[Secondary transurethral surgery of the bladder neck]. / Transurethrale Zweiteingriffe am Blasenhals.

In the period from 1970 to 1978 24 patients after transvesical adenomectomy and 76 patients after TUR of the prostate gland underwent a secondary transurethral surgical intervention. Causes were residual adenomas, stenoses of the vesical neck and recidivations of the adenoma. Explanations are given for the differences in the course of the temporary coordination to the primary intervention. The preoperative situation concerning infections, problems of the operation technique and in the case of the TUR the qualification of the resectionists are mentioned. After the TUR as primary intervention clearly less carcinomas were proved in the electro-resectate in comparison to the transvesical adenomectomy.

[Priapism--clinical aspects and treatment]. / Priapismus--Klinik und Behandlung.

After references to definition, anatomic and physiologic foundations the author deals with the pathology of priapism. A clinical systematization of the diseases occurring together with priapism according to causal points of view is no more worth being advocated. It is completely unclarified to which extent the disease actually participates in priapism. After the description of the possibilities of a conservative medical treatment to the heparin and fibrinolysis therapy it is referred to the importance of the continuous drainage of the corpora cavernosa after Grayhack and Quackels. It is reported on two patients operated according to Grayhack. Results of surgical therapy reported in publications of the last time refer to an essential improvement of the prognosis of priapism.