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- Aspergillus fumigatus (2)
- Bone-marrow-transplantation (2)
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- Stammzelltransplantation (2)
- chemotherapy (2)
- multiple myeloma (2)
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- Akute myeloische Leukämie (1)
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- Medizinische Klinik und Poliklinik II (42) (remove)
Background: There is much evidence that T cells are strongly involved in the pathogenesis of localized and systemic forms of scleroderma (SSc). A dysbalance between FoxP3+ regulatory CD4+ T cells (Tregs) and inflammatory T-helper (Th) 17 cells has been suggested. Methods: The study aimed (1) to investigate the phenotypical and functional characteristics of Th17 and Tregs in SSc patients depending on disease manifestation (limited vs. diffuse cutaneous SSc, dcSSc) and activity, and (2) the transcriptional level and methylation status of Th17- and Treg-specific transcription factors. Results: There was a concurrent accumulation of circulating peripheral IL-17-producing CCR6+ Th cells and FoxP3+ Tregs in patients with dcSSc. At the transcriptional level, Th17- and Treg-associated transcription factors were elevated in SSc. A strong association with high circulating Th17 and Tregs was seen with early, active, and severe disease presentation. However, a diminished suppressive function on autologous lymphocytes was found in SSc-derived Tregs. Significant relative hypermethylation was seen at the gene level for RORC1 and RORC2 in SSc, particularly in patients with high inflammatory activity. Conclusions: Besides the high transcriptional activity of T cells, attributed to Treg or Th17 phenotype, in active SSc disease, Tregs may be insufficient to produce high amounts of IL-10 or to control proliferative activity of effector T cells in SSc. Our results suggest a high plasticity of Tregs strongly associated with the Th17 phenotype. Future directions may focus on enhancing Treg functions and stabilization of the Treg phenotype.
Multiple myeloma (MM) is a plasma cell disorder that is characterized by a great genetic heterogeneity. Recent next generation sequencing studies revealed an accumulation of tumor-associated mutations in receptor tyrosine kinases (RTKs) which may also contribute to the activation of survival pathways in MM. To investigate the clinical role of RTK-mutations in MM, we deep-sequenced the coding DNA-sequence of EGFR, EPHA2, ERBB3, IGF1R, NTRK1 and NTRK2 which were previously found to be mutated in MM, in 75 uniformly treated MM patients of the “Deutsche Studiengruppe Multiples Myelom”. Subsequently, we correlated the detected mutations with common cytogenetic alterations and clinical parameters. We identified 11 novel non-synonymous SNVs or rare patient-specific SNPs, not listed in the SNP databases 1000 genomes and dbSNP, in 10 primary MM cases. The mutations predominantly affected the tyrosine-kinase and ligand-binding domains and no correlation with cytogenetic parameters was found. Interestingly, however, patients with RTK-mutations, specifically those with rare patient-specific SNPs, showed a significantly lower overall, event-free and progression-free survival. This indicates that RTK SNVs and rare patient-specific RTK SNPs are of prognostic relevance and suggests that MM patients with RTK-mutations could potentially profit from treatment with RTK-inhibitors.
Peptide Receptor Radionuclide Therapy (PRRT) for the treatment of neuroendocrine tumors may lead to kidney deterioration. This study aimed to evaluate the suitability of \(^{99m}\)Tc-mercaptoacetyltriglycine (\(^{99m}\)Tc-MAG3) clearance for the early detection of PRRT-induced changes on tubular extraction (TE). TE rate (TER) was measured prior to 128 PRRT cycles (7.6±0.4 GBq \(^{177}\)Lu-octreotate/octreotide each) in 32 patients. TER reduction during PRRT was corrected for age-related decrease and analyzed for the potential to predict loss of glomerular filtration (GF). The GF rate (GFR) as measure for renal function was derived from serum creatinine. The mean TER was 234 ± 53 ml/min/1.73 m² before PRRT (baseline) and 221 ± 45 ml/min/1.73 m² after a median follow-up of 370 days. The age-corrected decrease (mean: -3%, range: -27% to +19%) did not reach significance (p=0.09) but significantly correlated with the baseline TER (Spearman p=-0.62, p<0.001). Patients with low baseline TER showed an improved TER after PRRT, high decreases were only observed in individuals with high baseline TER. Pre-therapeutic TER data were inferior to plasma creatinine-derived GFR estimates in predicting late nephropathy. TER assessed by \(^{99m}\)Tc-MAG3clearance prior to and during PRRT is not suitable as early predictor of renal injury and an increased risk for late nephropathy.
Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Krüppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation.
Molecular changes in hepatic metabolism and transport in cirrhosis and their functional importance
(2016)
Liver cirrhosis is the common endpoint of many hepatic diseases and represents a relevant risk for liver failure and hepatocellular carcinoma. The progress of liver fibrosis and cirrhosis is accompanied by deteriorating liver function. This review summarizes the regulatory and functional changes in phase I and phase II metabolic enzymes as well as transport proteins and provides an overview regarding lipid and glucose metabolism in cirrhotic patients. Interestingly, phase I enzymes are generally downregulated transcriptionally, while phase II enzymes are mostly preserved transcriptionally but are reduced in their function. Transport proteins are regulated in a specific way that resembles the molecular changes observed in obstructive cholestasis. Lipid and glucose metabolism are characterized by insulin resistance and catabolism, leading to the disturbance of energy expenditure and wasting. Possible non-invasive tests, especially breath tests, for components of liver metabolism are discussed. The heterogeneity and complexity of changes in hepatic metabolism complicate the assessment of liver function in individual patients. Additionally, studies in humans are rare, and species differences preclude the transferability of data from rodents to humans. In clinical practice, some established global scores or criteria form the basis for the functional evaluation of patients with liver cirrhosis, but difficult treatment decisions such as selection for transplantation or resection require further research regarding the application of existing non-invasive tests and the development of more specific tests.
Tyrosine kinase inhibitors represent today's treatment of choice in chronic myeloid leukemia (CML). Allogeneic hematopoietic stem cell transplantation (HSCT) is regarded as salvage therapy. This prospective randomized CML-study IIIA recruited 669 patients with newly diagnosed CML between July 1997 and January 2004 from 143 centers. Of these, 427 patients were considered eligible for HSCT and were randomized by availability of a matched family donor between primary HSCT (group A; N = 166 patients) and best available drug treatment (group B; N = 261). Primary end point was long-term survival. Survival probabilities were not different between groups A and B (10-year survival: 0.76 (95% confidence interval (CI): 0.69-0.82) vs 0.69 (95% CI: 0.61-0.76)), but influenced by disease and transplant risk. Patients with a low transplant risk showed superior survival compared with patients with high( P < 0.001) and non-high-risk disease (P = 0.047) in group B; after entering blast crisis, survival was not different with or without HSCT. Significantly more patients in group A were in molecular remission (56% vs 39%; P = 0.005) and free of drug treatment (56% vs 6%; P < 0.001). Differences in symptoms and Karnofsky score were not significant. In the era of tyrosine kinase inhibitors, HSCT remains a valid option when both disease and transplant risk are considered.
Background
A phase I/II study and subsequent phase III study (MPACT) reported significant correlations between CA19-9 decreases and prolonged overall survival (OS) with nab-paclitaxel plus gemcitabine (nab-P + Gem) treatment for metastatic pancreatic cancer (MPC). CA19-9 changes at week 8 and potential associations with efficacy were investigated as part of an exploratory analysis in the MPACT trial.
Patients and methods
Untreated patients with MPC (N = 861) received nab-P + Gem or Gem alone. CA19-9 was evaluated at baseline and every 8 weeks.
Results
Patients with baseline and week-8 CA19-9 measurements were analyzed (nab-P + Gem: 252; Gem: 202). In an analysis pooling the treatments, patients with any CA19-9 decline (80%) versus those without (20%) had improved OS (median 11.1 versus 8.0 months; P = 0.005). In the nab-P + Gem arm, patients with (n = 206) versus without (n = 46) any CA19-9 decrease at week 8 had a confirmed overall response rate (ORR) of 40% versus 13%, and a median OS of 13.2 versus 8.3 months (P = 0.001), respectively. In the Gem-alone arm, patients with (n = 159) versus without (n = 43) CA19-9 decrease at week 8 had a confirmed ORR of 15% versus 5%, and a median OS of 9.4 versus 7.1 months (P = 0.404), respectively. In the nab-P + Gem and Gem-alone arms, by week 8, 16% (40/252) and 6% (13/202) of patients, respectively, had an unconfirmed radiologic response (median OS 13.7 and 14.7 months, respectively), and 79% and 84% of patients, respectively, had stable disease (SD) (median OS 11.1 and 9 months, respectively). Patients with SD and any CA19-9 decrease (158/199 and 133/170) had a median OS of 13.2 and 9.4 months, respectively.
Conclusion
This analysis demonstrated that, in patients with MPC, any CA19-9 decrease at week 8 can be an early marker for chemotherapy efficacy, including in those patients with SD. CA19-9 decrease identified more patients with survival benefit than radiologic response by week 8.
Comparison of nonculture blood-based tests for diagnosing invasive aspergillosis in an animal model
(2016)
The European Aspergillus PCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated "in vivo" specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and beta-D-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and beta-D-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and beta-D-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and beta-D-glucan proved optimal (area under the curve AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.
Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the "European Network for the Study of Cholangiocarcinoma" (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted.
Infections of the central nervous system (CNS) are infrequently diagnosed in immunocompetent patients, but they do occur in a significant proportion of patients with hematological disorders. In particular, patients undergoing allogeneic hematopoietic stem-cell transplantation carry a high risk for CNS infections of up to 15%. Fungi and Toxoplasma gondii are the predominant causative agents. The diagnosis of CNS infections is based on neuroimaging, cerebrospinal fluid examination and biopsy of suspicious lesions in selected patients. However, identification of CNS infections in immunocompromised patients could represent a major challenge since metabolic disturbances, side-effects of antineoplastic or immunosuppressive drugs and CNS involvement of the underlying hematological disorder may mimic symptoms of a CNS infection. The prognosis of CNS infections is generally poor in these patients, albeit the introduction of novel substances (e.g. voriconazole) has improved the outcome in distinct patient subgroups. This guideline has been developed by the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Medical Oncology (DGHO) with the contribution of a panel of 14 experts certified in internal medicine, hematology/oncology, infectious diseases, intensive care, neurology and neuroradiology. Grades of recommendation and levels of evidence were categorized by using novel criteria, as recently published by the European Society of Clinical Microbiology and Infectious Diseases.
The interleukin (IL)-12-type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor beta 1 (IL-12R beta 1) as one component of their receptor signaling complexes, with IL-12R beta 2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12R beta 1, whereas Jak2 binds to IL-23R and also to IL-12R beta 2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12R beta 1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12- and IL-23-induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12R beta 1 and Jak2 by IL-23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable.
Donor CD4\(^+\)Foxp3\(^+\) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2-and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.
Infectious complications after allogeneic haematopoietic stem cell transplantation (allo-HCT) remain a clinical challenge. This is a guideline provided by the AGIHO (Infectious Diseases Working Group) of the DGHO (German Society for Hematology and Medical Oncology). A core group of experts prepared a preliminary guideline, which was discussed, reviewed, and approved by the entire working group. The guideline provides clinical recommendations for the preventive management including prophylactic treatment of viral, bacterial, parasitic, and fungal diseases. The guideline focuses on antimicrobial agents but includes recommendations on the use of vaccinations. This is the updated version of the AGHIO guideline in the field of allogeneic haematopoietic stem cell transplantation utilizing methods according to evidence-based medicine criteria.
Objective
To analyse the role of multibiomarker disease activity (MBDA) score in predicting disease relapses in patients with rheumatoid arthritis (RA) in sustained remission who tapered disease modifying antirheumatic drug (DMARD) therapy in RETRO, a prospective randomised controlled trial.
Methods
MBDA scores (scale 1-100) were determined based on 12 inflammation markers in baseline serum samples from 94 patients of the RETRO study. MBDA scores were compared between patients relapsing or remaining in remission when tapering DMARDs. Demographic and disease-specific parameters were included in multivariate logistic regression analysis for defining predictors of relapse.
Results
Moderate-to-high MBDA scores were found in 33% of patients with RA overall. Twice as many patients who relapsed (58%) had moderate/high MBDA compared with patients who remained in remission (21%). Baseline MBDA scores were significantly higher in patients with RA who were relapsing than those remaining in stable remission (N=94; p=0.0001) and those tapering/stopping (N=59; p=0.0001). Multivariate regression analysis identified MBDA scores as independent predictor for relapses in addition to anticitrullinated protein antibody (ACPA) status. Relapse rates were low (13%) in patients who were MBDA-/ACPA-, moderate in patients who were MBDA+/ACPA- (33.3%) and MBDA-ACPA+ (31.8%) and high in patients who were MBDA+/ACPA+ (76.4%).
Conclusions
MBDA improved the prediction of relapses in patients with RA in stable remission undergoing DMARD tapering. If combined with ACPA testing, MBDA allowed prediction of relapse in more than 80% of the patients.
Trial registration number EudraCT
2009-015740-42.
Panobinostat is an oral pan-histone deacetylase inhibitor developed by Novartis. Panobinostat acts via epigenetic modification and inhibition of the aggresome pathway. In August 2015, the European Commission authorized panobinostat for use in combination with bortezomib and dexamethasone for the treatment of relapsed or relapsed and refractory multiple myeloma (MM) in patients who have received aeyen2 prior regimens including bortezomib and an immunomodulatory drug. In January 2016, the National Institute for Health and Care Excellence recommended panobinostat for use in the same combination and patient population. The authorization and recommendation were based on results from the pivotal phase 3 PANORAMA 1 (NCT01023308) clinical trial, which demonstrated an improvement in median progression-free survival of 7.8 months for the three-drug combination compared with placebo plus bortezomib and dexamethasone in this patient population. This review will discuss the current treatment landscape for relapsed/refractory MM, the mechanism of action of panobinostat, clinical data supporting the European authorization, concerns about safety and strategies for mitigating toxicity, and how panobinostat fits into the current MM landscape in Europe.
Objective
To use statistical methods to establish a threshold for individual response in patient-reported outcomes (PROs) in patients with rheumatoid arthritis.
Methods
We used an analysis of variance model in patients on stable therapy (discovery cohort) to establish critical differences (d(crit)) for the minimum change associated with a significant individual patient response (beyond normal variation) in the PRO measures of pain (0-10), fatigue (0-10), and function (Funktionsfragebogen Hannover questionnaire; 0-100). We then evaluated PRO responses in patients initiating adalimumab in a noninterventional study (treatment cohort).
Results
In the discovery cohort (n=700), PROs showed excellent long-term retest reliability. The minimum change that exceeded random fluctuation was conservatively determined to be 3 points for pain, 4 points for fatigue, and 16 points for function. In the treatment cohort (n=2,788), 1,483 patients (53.2%) achieved a significant individual therapeutic response as assessed by Disease Activity Score in 28 joints (DAS28)-d(crit) (1.8 points) after 12 months of adalimumab treatment; 68.5% of patients with a DAS28-d(crit) response achieved a significant improvement in pain, whereas approximately 40% achieved significant improvements in fatigue or function. Significant improvements in all 3 PROs occurred in 22.7% of patients; 22.8% did not have any significant PRO responses. In contrast, significant improvements in all 3 PROs occurred in only 4.4% of 1,305 patients who did not achieve a DAS28-d(crit) response at month 12, and 59.1% did not achieve any significant PRO responses.
Conclusion
The establishment of critical differences in PROs distinguishes true responses from random variation and provides insights into appropriate patient management.
Invasive Aspergillosis (IA) is an opportunistic infection caused by Aspergillus, a ubiquitously present airborne pathogenic mold. A growing number of studies suggest a major host genetic component in disease susceptibility. Here, we evaluated whether 14 single-nucleotide polymorphisms within NFκB1, NFκB2, RelA, RelB, Rel, and IRF4 genes influence the risk of IA in a population of 834 high-risk patients (157 IA and 677 non-IA) recruited through a collaborative effort involving the aspBIOmics consortium and four European clinical institutions. No significant overall associations between selected SNPs and the risk of IA were found in this large cohort. Although a hematopoietic stem cell transplantation (HSCT)-stratified analysis revealed that carriers of the IRF4rs12203592T/T genotype had a six-fold increased risk of developing the infection when compared with those carrying the C allele (ORREC = 6.24, 95%CI 1.25–31.2, P = 0.026), the association of this variant with IA risk did not reach significance at experiment-wide significant threshold. In addition, we found an association of the IRF4AATC and IRF4GGTC haplotypes (not including the IRF4rs12203592T risk allele) with a decreased risk of IA but the magnitude of the association was similar to the one observed in the single-SNP analysis, which indicated that the haplotypic effect on IA risk was likely due to the IRF4rs12203592 SNP. Finally, no evidence of significant interactions among the genetic markers tested and the risk of IA was found. These results suggest that the SNPs on the studied genes do not have a clinically relevant impact on the risk of developing IA.
Background
Over the past two decades, there has been a rising trend in malignant melanoma incidence worldwide. In 2008, Germany introduced a nationwide skin cancer screening program starting at age 35. The aims of this study were to analyse the distribution of malignant melanoma tumour stages over time, as well as demographic and regional differences in stage distribution and survival of melanoma patients.
Methods
Pooled data from 61 895 malignant melanoma patients diagnosed between 2002 and 2011 and documented in 28 German population-based and hospital-based clinical cancer registries were analysed using descriptive methods, joinpoint regression, logistic regression and relative survival.
Results
The number of annually documented cases increased by 53.2% between 2002 (N = 4 779) and 2011 (N = 7 320). There was a statistically significant continuous positive trend in the proportion of stage UICC I cases diagnosed between 2002 and 2011, compared to a negative trend for stage UICC II. No trends were found for stages UICC III and IV respectively. Age (OR 0.97, 95% CI 0.97–0.97), sex (OR 1.18, 95% CI 1.11–1.25), date of diagnosis (OR 1.05, 95% CI 1.04–1.06), ‘diagnosis during screening’ (OR 3.24, 95% CI 2.50–4.19) and place of residence (OR 1.23, 95% CI 1.16–1.30) had a statistically significant influence on the tumour stage at diagnosis. The overall 5-year relative survival for invasive cases was 83.4% (95% CI 82.8–83.9%).
Conclusions
No distinct changes in the distribution of malignant melanoma tumour stages among those aged 35 and older were seen that could be directly attributed to the introduction of skin cancer screening in 2008.
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A complex aberrant karyotype consisting of multiple unrelated cytogenetic abnormalities is associated with poor prognosis in patients with acute myeloid leukemia (AML). The European Leukemia Net classification and the UK Medical Research Council recommendation provide prognostic categories that differ in the definition of unbalanced aberrations as well as the number of single aberrations. The aim of this study on 3526 AML patients was to redefine and validate a cutoff for karyotype complexity in AML with regard to adverse prognosis. Our study demonstrated that (1) patients with a pure hyperdiploid karyotype have an adverse risk irrespective of the number of chromosomal gains, (2) patients with translocation t(9;11)(p21∼22;q23) have an intermediate risk independent of the number of additional aberrations, (3) patients with 4 abnormalities have an adverse risk per se and (4) patients with three aberrations in the absence of abnormalities of strong influence (hyperdiploid karyotype, t(9;11)(p21∼22;q23), CBF-AML, unique adverse-risk aberrations) have borderline intermediate/adverse risk with a reduced overall survival compared with patients with a normal karyotype.
We compared outcomes from a single-arm study of blinatumomab in adult patients with B-precursor Ph-negative relapsed/refractory acute lymphoblastic leukemia (R/R ALL) with a historical data set from Europe and the United States. Estimates of complete remission (CR) and overall survival (OS) were weighted by the frequency distribution of prognostic factors in the blinatumomab trial. Outcomes were also compared between the trial and historical data using propensity score methods. The historical cohort included 694 patients with CR data and 1112 patients with OS data compared with 189 patients with CR and survival data in the blinatumomab trial. The weighted analysis revealed a CR rate of 24% (95% CI: 20–27%) and a median OS of 3.3 months (95% CI: 2.8–3.6) in the historical cohort compared with a CR/CRh rate of 43% (95% CI: 36–50%) and a median OS of 6.1 months (95% CI: 4.2–7.5) in the blinatumomab trial. Propensity score analysis estimated increased odds of CR/CRh (OR=2.68, 95% CI: 1.67–4.31) and improved OS (HR=0.536, 95% CI: 0.394–0.730) with blinatumomab. The analysis demonstrates the application of different study designs and statistical methods to compare novel therapies for R/R ALL with historical data.
Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.
Hsp90 inhibition ameliorates CD4\(^{+}\) T cell-mediated acute Graft versus Host disease in mice
(2016)
Introduction:
For many patients with leukemia only allogeneic bone marrow transplantion provides a chance of cure. Co‐transplanted mature donor T cells mediate the desired Graft versus Tumor (GvT) effect required to destroy residual leukemic cells. The donor T cells very often, however, also attack healthy tissue of the patient inducing acute Graft versus Host Disease (aGvHD)—a potentially life‐threatening complication.
Methods:
Therefore, we used the well established C57BL/6 into BALB/c mouse aGvHD model to evaluate whether pharmacological inhibition of heat shock protein 90 (Hsp90) would protect the mice from aGvHD.
Results:
Treatment of the BALB/c recipient mice from day 0 to +2 after allogeneic CD4\(^{+}\) T cell transplantation with the Hsp90 inhibitor 17‐(dimethylaminoethylamino)‐17‐demethoxygeldanamycin (DMAG) partially protected the mice from aGvHD. DMAG treatment was, however, insufficient to prolong overall survival of leukemia‐bearing mice after transplantation of allogeneic CD4\(^{+}\) and CD8\(^{+}\) T cells. Ex vivo analyses and in vitro experiments revealed that DMAG primarily inhibits conventional CD4\(^{+}\) T cells with a relative resistance of CD4\(^{+}\) regulatory and CD8\(^{+}\) T cells toward Hsp90 inhibition.
Conclusions:
Our data, thus, suggest that Hsp90 inhibition might constitute a novel approach to reduce aGvHD in patients without abrogating the desired GvT effect.
Ampullary carcinoma is a rare tumor and evidence on the treatment of recurrent metastatic disease is scarce. We report the case of a 60-year-old patient with an R0-resected node-positive adenocarcinoma of the papilla of Vater of an initially diagnosed intestinal subtype who developed pulmonary metastases 2 months after adjuvant gemcitabine chemotherapy and, subsequently, liver metastases. Palliative combination chemotherapy with standard regimens for intestinal-type adenocarcinoma (FOLFOX and FOLFIRI) failed. However, subsequent combination chemotherapy with nanoparticle albumin-bound paclitaxel and gemcitabine, a regimen with proven efficacy in metastatic adenocarcinoma of the pancreas, resulted in a durable, very good partial remission. Treatment was manageable and well tolerated. Primary tumor and metastatic tissue were reassessed by immunohistochemistry and had to be reclassified to a mixed phenotype containing predominant elements of the pancreatobiliary subtype. Our case suggests that combination chemotherapy with nanoparticle albumin-bound paclitaxel and gemcitabine could represent a promising option for the treatment of this rare disease and warrants further investigation within controlled clinical trials. Moreover, thorough characterization of ampullary carcinomas by histomorphology and additional immunohistochemistry should become mandatory in order to start a chemotherapeutic regimen tailored for the definitive subtype.
Introduction
Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.
Results
We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.
Conclusion
We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.
Background
Ribavirin blood levels vary considerably between patients with standard weight-based dosing. Their impact on sustained virological response (SVR) with pegylated interferon and ribavirin is controversial, but has mostly been studied before the IL28b gene polymorphism as a possible confounder was discovered.
Methods
The impact of serum ribavirin trough levels at week 4, at the end of treatment and of mean levels across the entire antiviral treatment with pegylated interferon and ribavirin on relapse, SVR rates and anemia was retrospectively studied by univariate and multivariable logistic regression analyses in 214 patients with HCV genotype 1–4 infection, including 88 patients with available IL28b genotyping.
Results
Mean ribavirin levels varied between 0.68–5.65 mg/l and significantly differed between patients with or without SVR. By multivariable regression including age, sex, HCV viral load, HCV genotype, liver fibrosis stage, prior treatments, immunosuppression and IL28b genotype, ribavirin levels consistently displayed significant influence on SVR and relapse without indication for a specific importance of higher concentrations early or late in the treatment course. Although hemoglobin decline was on average more pronounced in patients with higher ribavirin levels, hemoglobin remained relatively stable in a significant proportion of these, indicating that ribavirin levels alone are insufficient to predict anemia.
Conclusion
While data are scarce to draw conclusions applicable for modern DAA therapies, these results support ribavirin treatment based on serum levels instead of purely weight-based dosing in combination with pegylated interferon.
Background
In adult patients with Duchenne muscular dystrophy (DMD) life-threatening constipation has been reported. Since gastrointestinal function in DMD has not been rigorously studied we investigated objective and subjective manifestations of gastrointestinal disturbances in DMD patients.
Methods
In 33 patients with DMD, age 12–41 years, eating behavior and gastrointestinal symptoms were evaluated by questionnaires. Gastric emptying half time (T\(_{1/2}\)) and oro-cecal transit time (OCTT) were evaluated by analyzing \(^{13}\)CO\(_{2}\) exhalation curves after ingestion of \(^{13}\)C labeled test meals. Colonic transit time (CTT) was measured by abdominal radiography following ingestion of radiopaque markers.
Results
The median (quartiles) T\(_{1/2}\) was 187 (168, 220) minutes, the OCTT was 6.3 (5.0, 7.9) hours, both substantially longer than normal data (Goetze 2005, T\(_{1/2}\): 107±10; Geypens 1999, OCTT 4.3±0.1 hours). The median CTT was 60 (48, 82) hours despite extensive use of laxative measures (Meier 1995, upper limit of normal: 60 hours). T\(_{1/2}\) and OCTT did not correlate with symptoms evaluated by the Gastroparesis Cardinal Symptom Index (GCSI) (Spearman r = -0.3, p = 0.1; and r = -0.15, p = 0.4, respectively). CTT was not correlated with symptoms of constipation assessed by ROME III criteria (r = 0.12, p = 0.5).
Conclusions
DMD patients have a markedly disturbed gastrointestinal motor function. Since objective measures of impaired gastrointestinal transport are not correlated with symptoms of gastroparesis or constipation our findings suggest that measures assuring adequate intestinal transport should be taken independent of the patient’s perception in order to prevent potentially life threatening constipation, particularly in older DMD patients.
Invasive aspergillosis (IA) is a devastating opportunistic infection and its treatment constitutes a considerable burden for the health care system. Immunocompromised patients are at an increased risk for IA, which is mainly caused by the species Aspergillus fumigatus. An early and reliable diagnosis is required to initiate the appropriate antifungal therapy. However, diagnostic sensitivity and accuracy still needs to be improved, which can be achieved at least partly by the definition of new biomarkers. Besides the direct detection of the pathogen by the current diagnostic methods, the analysis of the host response is a promising strategy toward this aim. Following this approach, we sought to identify new biomarkers for IA. For this purpose, we analyzed gene expression profiles of hematological patients and compared profiles of patients suffering from IA with non-IA patients. Based on microarray data, we applied a comprehensive feature selection using a random forest classifier. We identified the transcript coding for the S100 calcium-binding protein B (S100B) as a potential new biomarker for the diagnosis of IA. Considering the expression of this gene, we were able to classify samples from patients with IA with 82.3% sensitivity and 74.6% specificity. Moreover, we validated the expression of S100B in a real-time reverse transcription polymerase chain reaction (RT-PCR) assay and we also found a down-regulation of S100B in A. fumigatus stimulated DCs. An influence on the IL1B and CXCL1 downstream levels was demonstrated by this S100B knockdown. In conclusion, this study covers an effective feature selection revealing a key regulator of the human immune response during IA. S100B may represent an additional diagnostic marker that in combination with the established techniques may improve the accuracy of IA diagnosis.
Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4\(^+\) or CD8\(^+\) T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b\(^+\) myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b\(^+\) myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions.
Die Funktion eines Genes zu erforschen, indem man es ausschaltet, und damit seiner Rolle im komplexen Zusammenspiel der einzelnen Prozesse des menschlichen Körpers nachzugehen, stellt heutzutage eines der vielversprechendsten Felder der Gentechnologie dar.
Der als RNA-Interferenz bekannte Mechanismus wurde in dieser Arbeit durch den Einsatz von sogenannten short hairpin RNAs (shRNAs), dauerhaft in das Wirtsgenom integrierter Knockdown-Träger, für das Gen bzw. Protein Y box binding Protein (YBX1) angewendet.
YBX1, ein Vertreter der Cold-Shock-Proteine, stellt einen zentralen Interakteur lebensnotwendiger Prozesse wie Proliferation, Apoptose und Embryogenese im menschlichen Körper dar. Seine Dysregulation wird jedoch auch in einen Zusammenhang mit Entzündung, Tumorformation und –aufrechterhaltung gebracht, unter anderem auch für das Multiple Myelom.
Das Multiple Myelom ist für 1% aller Krebserkrankungen weltweit verantwortlich mit noch immer ungelösten Problemen unzulänglicher Therapie und deletärer Prognose.
In der vorliegenden Arbeit wurde die Möglichkeit geschaffen, die Rolle von YBX1 für das Multiple Myelom mit Hilfe einer Maus-Plasmazelllinie in vivo zu untersuchen. Dies geschah durch die Suppression der Genexpression von YBX1 mittels verschiedener gegen YBX1 gerichteter Polymerase II-getriebener short hairpin RNAs (shRNAs). Diese wurden in ein lentivirales Plasmid kloniert. Durch das Vorhandensein von Tetrazyklin induzierbaren Promotoren (Tet-On bzw. Tet-Off) wurde die Möglichkeit geschaffen, einen konditionellen Knockdown von YBX1 zu induzieren. Dies war notwendig, da initiale Arbeiten mit humanen Myelomzelllinien zeigten, dass der Knockdown von YBX1 Apoptose induzieren kann. Mit diesem Konstrukt wurden in HEK293 Zellen lentivirale Partikel hergestellt und damit die murine Plasmozytomzelle MOPC315.BM stabil transduziert. Nach Selektion, Klonierung und Testung (Puromycin-Selektion, RFP-Expression und Western-Blot Analyse) stand ein Zellklon zur Verfügung, der einen induzierbaren YBX1 Knockdown zeigt.
Damit gelang die Etablierung eines gegen YBX1 gerichteten Vektorsystems in einer murinen Plasmazellinie in vitro. Mit Hilfe dieser Zelllinien kann nun in weiteren Arbeiten untersucht werden, wie ein YBX1 Knockdown das Tumorwachstum in vivo beinflusst.
Background
Anastomotic leakage (AL) is one of the most common and serious complications following visceral surgery. In recent years, endoluminal vacuum therapy has dramatically changed therapeutic options for AL, but its use has been limited to areas easily accessible by endoscope.
Case presentation
We describe the first use of endoluminal vacuum therapy in the small intestine employing a combined surgical and endoscopic “rendezvous technique” in which the surgeon assists the endoscopic placement of an endoluminal vacuum therapy sponge in the jejunum by means of a pullback string. This technique led to a completely closed AL after 27 days and 7 changes of the endosponge.
Conclusion
The combined surgical and endoscopic rendezvous technique can be useful in cases of otherwise difficult endosponge placement.
Wilms tumor protein 1 (WT1) is a suitable target to develop an immunotherapeutic approach against high risk acute myeloid leukemia (AML), particularly their relapse after allogeneic hematopoietic stem cell transplantation (HSCT). As an intracellular protein traversing between nucleus and cytoplasm, recombinant expression of WT1 is difficult. Therefore, an induction of WT1-specific T-cell responses is mostly based on peptide vaccination as well as dendritic cell (DC) electroporation with mRNA encoding full-length protein to mount WT1-derived peptide variations presented to T cells. Alternatively, the WT1 peptide presentation could be broadened by forcing receptor-mediated endocytosis of DCs.
In this study, antibody fusion proteins consisting of an antibody specific to the human DEC205 endocytic receptor and various fragments of WT1 (anti-hDEC205-WT1) were generated for a potential DC-targeted recombinant WT1 vaccine. Anti-hDEC205-WT1 antibody fusion proteins containing full-length or major parts of WT1 were not efficiently expressed and secreted due to their poor solubility and secretory capacity. However, small fragment-containing variants: anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were obtained in good yields.
Since three of these fusion proteins contain the most of the known immunogenic epitopes in their sequences, the anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were tested for their T-cell stimulatory capacities. Mature monocyte-derived DCs loaded with anti-hDEC205-WT191-138 could induce ex vivo T-cell responses in 12 of 16 blood samples collected from either healthy or HSC transplanted individuals compared to included controls (P < 0.01). Furthermore, these T cells could kill WT1-overexpressing THP-1 leukemia cells in vitro after expansion.
In conclusion, alongside proving the difficulty in expression and purification of intracellular WT1 as a vaccine protein, our results from this work introduce an alternative therapeutic vaccine approach to improve an anti-leukemia immune response in the context of allogeneic HSCT and potentially beyond.
Hintergrund
In Südafrika sind ca. 30 % aller schwangeren Frauen mit HIV infiziert. Physiologische Veränderungen in der Schwangerschaft alterieren die Metabolisierung der antiretroviralen Medikamente, was subtherapeutische oder toxische Serumspiegel zur Folge haben kann. Konsequenzen ergeben sich in Form einer Risikoerhöhung einer Mutter-Kind-Übertragung und Resistenzentwicklung einerseits sowie toxischer Wirkungen andererseits. Über die Pharmakokinetik antiretroviraler Substanzen bei Schwangeren ist bislang wenig bekannt. In der vorliegenden Untersuchung wurden deshalb im Setting der routinemäßigen Patientenversorgung Nevirapin (NVP)- und Efavirenz (EFV)-Serumkonzentrationen bei antiretroviral behandelten Schwangeren gemessen.
Methoden
Die Untersuchungen erfolgten im Rahmen des DFG-geförderten Internationalen Graduiertenkollegs IRTG1522, einem Partnerprojekt der Universitäten Stellenbosch und Kapstadt/Südafrika und Würzburg, prospektiv im Tygerberg Academic Hospital in Kapstadt. Bei 108 HIV-infizierten Schwangeren, die mindestens seit 4 Wochen eine NVP- (n=52) oder EFV-Medikation (n=56) erhielten, wurden Serumproben entnommen. Die Messung der NVP- und EFV-Serumkonzentrationen erfolgte im TDM-Labor der Infektiologie am Universitätsklinikum Würzburg mittels einer etablierten Gaschromatographie bzw. Hochflüssigkeitschromatographie-Methode.
Ergebnisse
Nur 28 Proben (54%) der NVP-Konzentrationen lagen im therapeutischen Bereich (3.000 - 8.000 ng/ml) 36 Proben (64%) der EFV-Konzentrationen lagen im therapeutischen Bereich (1.000 - 4.000 ng/ml). Es zeigte sich ein signifikanter negativer Zusammenhang zwischen EFV-Konzentrationen und einem BMI >25.
In den univariaten Regressionen der einzelnen Variablen gab es einen signifikanten negativen Zusammenhang zwischen NVP-Konzentrationen und der Anzahl an Schwangerschaftswochen sowie einen signifikant positiven Zusammenhang zwischen NVP-Konzentrationen bei einer Begleitmedikation mit Valproat.
Es konnte ein signifikanter Zusammenhang zwischen NVP-Konzentrationen <3.000 ng/ml und einem virologischen Versagen nachgewiesen werden. Bei EFV konnte dieser signifikante Zusammenhang zu Konzentrationen <1.000 ng/ml gefunden werden.
Mittels Therapeutischem Drug Monitoring konnten 6 NVP- und 4 EFV-Serumspiegel detektiert werden, die eine unzureichende Medikamenteneinnahme der Patientinnen widerspiegelten. Bei einer Patientin fand eine vertikale Transmission statt.
Mit dieser Untersuchung konnte gezeigt werden, dass eine große Anzahl an schwangeren Frauen Serumspiegel außerhalb des therapeutischen Bereiches aufwiesen. Die pharmakokinetischen Veränderungen im Laufe der Schwangerschaft waren am ehesten Ursache für subtherapeutische Serumspiegel. Weiterhin zeigten sich subtherapeutische Konzentrationen als Auslöser von virologischem Versagen.
Schlussfolgerungen
Die Ergebnisse dieser Untersuchung legen nahe, dass ein Therapeutisches Drug Monitoring integraler Bestandteil der antiretroviralen Therapie von schwangeren Frauen im dritten Trimenon in Südafrika sein sollte.
Background
Multimodal treatment strategies – perioperative chemotherapy (CTx) and radical surgery – are currently accepted as treatment standard for locally advanced gastric cancer. However, the role of adjuvant postoperative CTx (postCTx) in addition to neoadjuvant preoperative CTx (preCTx) in this setting remains controversial.
Methods
Between 4/2006 and 12/2013, 116 patients with locally advanced gastric cancer were treated with preCTx. 72 patients (62 %), in whom complete tumor resection (R0, subtotal/total gastrectomy with D2-lymphadenectomy) was achieved, were divided into two groups, one of which receiving adjuvant therapy (n = 52) and one without (n = 20). These groups were analyzed with regard to survival and exclusion criteria for adjuvant therapy.
Results
Postoperative complications, as well as their severity grade, did not correlate with fewer postCTx cycles administered (p = n.s.). Long-term survival was shorter in patients receiving postCTx in comparison to patients without postCTx, but did not show statistical significance. In per protocol analysis by excluding two patients with perioperative death, a shorter 3-year survival rate was observed in patients receiving postCTx compared to patients without postCTx (3-year survival: 71.2 % postCTx group vs. 90.0 % non-postCTx group; p = 0.038).
Conclusion
These results appear contradicting to the anticipated outcome. While speculative, they question the value of post-CTx. Prospectively randomized studies are needed to elucidate the role of postCTx.
Role of PTEN in Oxidative Stress and DNA Damage in the Liver of Whole-Body Pten Haplodeficient Mice
(2016)
Type 2 diabetes (T2DM) and obesity are frequently associated with non-alcoholic fatty liver disease (NAFLD) and with an elevated cancer incidence. The molecular mechanisms of carcinogenesis in this context are only partially understood. High blood insulin levels are typical in early T2DM and excessive insulin can cause elevated reactive oxygen species (ROS) production and genomic instability. ROS are important for various cellular functions in signaling and host defense. However, elevated ROS formation is thought to be involved in cancer induction. In the molecular events from insulin receptor binding to genomic damage, some signaling steps have been identified, pointing at the PI3K/AKT pathway. For further elucidation Phosphatase and Tensin homolog (Pten), a tumour suppressor phosphatase that plays a role in insulin signaling by negative regulation of PI3K/AKT and its downstream targets, was investigated here. Dihydroethidium (DHE) staining was used to detect ROS formation in immortalized human hepatocytes. Comet assay and micronucleus test were performed to investigate genomic damage in vitro. In liver samples, DHE staining and western blot detection of HSP70 and HO-1 were performed to evaluate oxidative stress response. DNA double strand breaks (DSBs) were detected by immunohistostaining. Inhibition of PTEN with the pharmacologic inhibitor VO-OHpic resulted in increased ROS production and genomic damage in a liver cell line. Knockdown of Pten in a mouse model yielded increased oxidative stress levels, detected by ROS levels and expression of the two stress-proteins HSP70 and HO-1 and elevated genomic damage in the liver, which was significant in mice fed with a high fat diet. We conclude that PTEN is involved in oxidative stress and genomic damage induction in vitro and that this may also explain the in vivo observations. This further supports the hypothesis that the PI3K/AKT pathway is responsible for damaging effects of high levels of insulin.
The KISS1 Receptor as an In Vivo Microenvironment Imaging Biomarker of Multiple Myeloma Bone Disease
(2016)
Multiple myeloma is one of the most common hematological diseases and is characterized by an aberrant proliferation of plasma cells within the bone marrow. As a result of crosstalk between cancer cells and the bone microenvironment, bone homeostasis is disrupted leading to osteolytic lesions and poor prognosis. Current diagnostic strategies for myeloma typically rely on detection of excess monoclonal immunoglobulins or light chains in the urine or serum. However, these strategies fail to localize the sites of malignancies. In this study we sought to identify novel biomarkers of myeloma bone disease which could target the malignant cells and/or the surrounding cells of the tumor microenvironment. From these studies, the KISS1 receptor (KISS1R), a G-protein-coupled receptor known to play a role in the regulation of endocrine functions, was identified as a target gene that was upregulated on mesenchymal stem cells (MSCs) and osteoprogenitor cells (OPCs) when co-cultured with myeloma cells. To determine the potential of this receptor as a biomarker, in vitro and in vivo studies were performed with the KISS1R ligand, kisspeptin, conjugated with a fluorescent dye. In vitro microscopy showed binding of fluorescently-labeled kisspeptin to both myeloma cells as well as MSCs under direct co-culture conditions. Next, conjugated kisspeptin was injected into immune-competent mice containing myeloma bone lesions. Tumor-burdened limbs showed increased peak fluorescence compared to contralateral controls. These data suggest the utility of the KISS1R as a novel biomarker for multiple myeloma, capable of targeting both tumor cells and host cells of the tumor microenvironment.
Neben Infektionen und Graft-versus-Host-Reaktionen nach allogener Stammzelltransplantation, stellen das Rezidiv der Grunderkrankungen und die Transplantatabstoßung die schwerwiegendsten Probleme bei diesem Patientenklientel dar. Um jene frühzeitig zu erkennen, werden Chimärismusanalysen eingesetzt, mit deren Hilfe das Auftauchen kleinster Mengen an Empfängerknochenmarkszellen im peripheren Blut nachgewiesen werden können.
Hierfür stehen verschiedene Möglichkeiten mit unterschiedlichen Sensitivitäten und Anwendungsbereichen zur Verfügung, wie die Fluoreszenz-in-situ-Hybridisierung (FISH), die Amplifikation von short tandem repeats (STR) mittels Polymerasekettenreaktion (PCR) und die allelspezifische quantitative Real-time-PCR (qRT-PCR) mittels TaqMan, um die es in dieser Arbeit geht.
Mit Hilfe von speziellen Zielsequenzen auf unterschiedlichen Allelen, die Alizadeh et al. 2002 veröffentlichten, kann in der qRT-PCR bereits eine von 1000 Zellen nachgewiesen werden und somit zu einem frühen Zeitpunkt ein mögliches Rezidiv oder eine Abstoßung erkannt werden.
In dieser Arbeit wurden für die beschriebenen Allele und das SRY-Gen Standardreihen mit unterschiedlichen Konzentrationsstufen erstellt, mit Hilfe derer man die Ergebnisse der PCR aus Patientenproben einordnen und den Chimärismus berechnen konnte. Eine zusätzliche Kalibrierung der Proben wurde mit Standardreihen vorbestimmter Konzentrationsstufen des Housekeeping-Gens HCK durchgeführt, das auch bei der Auswertung der Patientenproben zum Einsatz kam.
Somit war es im Rahmen der Etablierung der PCR an der Uniklinik Würzburg möglich, in dieser Arbeit 395 Proben zu bestimmen, von denen 127 Proben von 26 Patienten ausgewertet und mit extern ermittelten STR-PCR-Ergebnissen verglichen werden konnten.
Die hieraus gewonnenen Daten wurden mit den von Alizadehet al.[59] veröffentlichten Daten verglichen bezüglich der Anwendbarkeit der allelspezifischen PCR auf das Patientenkollektiv der Uniklinik Würzburg und der Auswertung ihrer Sensitivität sowie klinischen Verwendbarkeit.
50
Um die ermittelten Chimärismen in einen klinischen Zusammenhang zu stellen, erfolgte die Zuordnung zu vier Gruppen mit verschiedenen Prozentspannen, bei denen unterschiedliche Szenarien in der klinischen Bewertung durchgespielt wurden.
Die Schwächen der etablierten PCR bestanden vor allem darin, dass 12,5% der Proben dieser Methode nicht zugänglich waren und angenommen werden muss, dass der Assay z.T. zu sensitiv war. Gerade in einem Bereich von > 5%igen Chimärismen stimmten die erhobenen Daten nicht mehr mit den Kontrollen überein, sondern gaben möglicherweise falsch hohe Chimärismen an.
Fehlende prospektive Daten machten es nicht möglich, in der Arbeit unstimmige Werte durch Beobachtung des weiteren klinischen Verlaufs auf ihre Richtigkeit zu prüfen.
Für die weitere Bewertung des Assays wäre es wichtig, dies in zukünftige Untersuchungen mit einzubeziehen.
Background
HIV-disease progression correlates with immune activation. Here we investigated whether corticosteroid treatment can attenuate HIV disease progression in antiretroviral-untreated patients.
Methods
Double-blind, placebo-controlled randomized clinical trial including 326 HIV-patients in a resource-limited setting in Tanzania (clinicaltrials.gov NCT01299948). Inclusion criteria were a CD4 count above 300 cells/μl, the absence of AIDS-defining symptoms and an ART-naïve therapy status. Study participants received 5 mg prednisolone per day or placebo for 2 years. Primary endpoint was time to progression to an AIDS-defining condition or to a CD4-count below 200 cells/μl.
Results
No significant change in progression towards the primary endpoint was observed in the intent-to-treat (ITT) analysis (19 cases with prednisolone versus 28 cases with placebo, p = 0.1407). In a per-protocol (PP)-analysis, 13 versus 24 study participants progressed to the primary study endpoint (p = 0.0741). Secondary endpoints: Prednisolone-treatment decreased immune activation (sCD14, suPAR, CD38/HLA-DR/CD8+) and increased CD4-counts (+77.42 ± 5.70 cells/μl compared to -37.42 ± 10.77 cells/μl under placebo, p < 0.0001). Treatment with prednisolone was associated with a 3.2-fold increase in HIV viral load (p < 0.0001). In a post-hoc analysis stratifying for sex, females treated with prednisolone progressed significantly slower to the primary study endpoint than females treated with placebo (ITT-analysis: 11 versus 21 cases, p = 0.0567; PP-analysis: 5 versus 18 cases, p = 0.0051): No changes in disease progression were observed in men.
Conclusions
This study could not detect any significant effects of prednisolone on disease progression in antiretroviral-untreated HIV infection within the intent-to-treat population. However, significant effects were observed on CD4 counts, immune activation and HIV viral load. This study contributes to a better understanding of the role of immune activation in the pathogenesis of HIV infection.
Background
Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation.
Objective
To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt\(^{-/-}\)) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke.
Results
In 5Htt\(^{-/-}\) platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca\(^{2+}\) entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt\(^{-/-}\) platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt\(^{-/-}\) mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt\(^{-/-}\) mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice.
Conclusion
Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization.
Background
Medulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup.
Methods
We established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma.
Results
Besides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture.
Conclusions
This model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.
Polymorphonuclear neutrophils (PMNs) are phagocytic cells of the innate immune system that efficiently kill bacteria. However, they also have regulatory effects on other immune cells and contribute to immunosuppression in cancer, which worsens the outcome. In particular, this has been demonstrated for a subset of granulocytic cells called myeloid- derived suppressor cells (MDSCs), but its distinction from PMNs is controversial. Most authors have explored the suppressive effects of MDSCs on T cells, but recent data suggest that NK cells are also affected. NK cells are crucial for the combat of tumor cells, in particular leukemic cells. There is hardly data available on the interaction between NK cells and suppressive granulocytic cells. Therefore, the aim of this thesis was to explore the effects of MDSCs and PMNs on the NK cell function against the leukemia cell line K562.
In co-culture experiments, I demonstrate that granulocytic MDSCs and PMNs had similar effects on NK cell function and homeostasis. On the one hand, they positively influenced the survival and maturation of NK cells. On the other, they inhibited the activation, cytotoxicity and cytokine production of NK cells, both IFNγ and TNFα, in response to K562 target cells. Furthermore, I show a down-regulation of the activating receptor NKp30 on NK cells in the presence of MDSCs or PMNs, which may form part of the underlying suppressive mechanisms.
However, there is also evidence for the involvement of other molecules. Further investigations are needed to confirm a relevant suppression of NK cells by granulocytic cells in cancer patients, and to identify therapeutic targets. The recognition that regular PMNs have similar effects on NK cells as MDSCs could simplify future experiments, since MDSCs are heterogeneous and laborious to isolate and identify.
NKcells and granulocytes are among the first immune cells to reconstitute after hematopoietic stem cell transplantation, and NK cells may be particularly exposed to suppressive effects of granulocytes this scenario. Modulating these suppressive effects of granulocytes on NK cells therapeutically may yield a better NK cell function and an improved cancer prognosis.

Ziel dieser Arbeit ist es, weitere Einblicke in die Aktivierung von FGF19 und
FXR durch diverse nukleäre Agonisten und deren spezifischer Rezeptoren zu
gewinnen. Hierbei soll im humanen Zellmodell versucht und mittels DNA-Analyse
untersucht werden, welche messbaren molekularbiologischen
Auswirkungen eine Behandlung mit unterschiedlichen Substanzen in
variierenden Konzentrationen bewirkt. Genauer soll betrachtet werden, ob sich
Vitamin A und Vitamin D als Induktoren von FGF19 in menschlichen
Darmzelllinien eignen, da dies bereits im Mausmodel demonstriert werden
konnte.
Dieser initialen Vermutung folgend, sollen auch die möglichen
Wechselwirkungen und Synergismen untersucht werden – welche
Mechanismen liegen diese zu Grunde und über welche molekularen
Signalwege werden dies vermuteten Effekte vermittelt.
Hierdurch soll ein besseres Verständnis für die Rezeptor und Agonistenabhängigen
Abläufe ermöglicht werden, um mögliche Rückschlüsse auf weitere
Funktionen bereits bekannter Vertreter zu erlauben.
Aufgrund der bereits oben beschriebenen Tiermodelle und der daraus
gewonnenen Einsichten würde sich durch ein noch besseres Verständnis des
FGF15/19 und des Farnesoid X Rezeptors in menschlichen Zellen, auf eine
zukünftige Anwendung in analytischen und/oder therapeutischen Bereichen
hoffen lassen.
Diese Arbeit soll sich deshalb den Fragen widmen, ob eine FGF19 Induktion in
humanen Darmzellen durch die nukleären Agonisten VD3, 9-cis RA und CDCA,
ähnlich dem Mausmodel, möglich ist und welche Faktoren dabei Einflüsse auf
die beschriebenen Effekte haben.
Opportunistische Infektionen stellen ein ernst zu nehmendes Problem bei immunsupprimierten Patienten in der Hämatologie und Onkologie sowie speziell bei Stammzell-transplantierten Patienten dar. Vor allem die Inzidenz invasiver Pilzinfektionen stieg in den letzten Jahrzehnten in diesem Risikokollektiv immer weiter an. Dabei verbleibt die Morbidität und Mortalität bei Patienten mit einer systemischen Mykose trotz verbesserter Diagnostik und Therapie weiterhin hoch.
Im Rahmen dieser Studie sollte die Inzidenz und Relevanz invasiver Mykosen am Standort Würzburg untersucht werden und die wesentlichen Risikofaktoren für eine Pilzinfektion bei hämatoonkologischen Hochrisiko-Patienten heraus gearbeitet werden. Um dies zu verwirklichen, wurden die Daten von 41 Patienten, welche eine allogene Stammzelltransplantation am Universitäts-klinikum Würzburg erhielten, detailliert dokumentiert und analysiert. Die aktive Beobachtung fand dabei bis 100 Tage nach Beginn der Konditionierungs-Chemotherapie statt. Eine Nachbetrachtung der Patientendaten in Bezug auf invasive Pilzinfektionen wurde ein halbes Jahr nach Studienstart durchgeführt.
Zur Diagnostik von invasiven Pilzinfektionen wurden im Beobachtungszeitraum bei allen Studienteilnehmern regelmäßig thorakale CT-Untersuchungen und Bestimmungen des Aspergillus-typischen Antigens Galactomannan im Serum durchgeführt. Zusätzlich erfolgte bei einem Teil der Patienten eine PCR-Untersuchung ihres Serums auf Aspergillus-DNA. Dabei zeigte die PCR- Untersuchung eine 100%ige Sensitivität und 55%ige Spezifität bei einem einmalig positiven Ergebnis. Wenn zwei positive PCR-Ergebnisse gefordert wurden, war die Sensitivität deutlich reduziert. Bei der Betrachtung der Galactomannan-Bestimmung ergab sich eine 74%ige Spezifität bei einem zweimalig positiven Ergebnis.
Von den 41 Studienteilnehmern erkrankten innerhalb des ersten halben Jahres nach der Konditionierung 22% an einer wahrscheinlichen invasiven Mykose und 17% an einer möglichen invasiven Mykose. Dabei waren 94% der Erkrankungen auf den Schimmelpilz Aspergillus spp. zurückzuführen. Lediglich bei einem Patienten wurde der Schlauchpilz Fusarium spp. nachgewiesen. Systemische Hefepilzinfektionen traten nicht auf. Alle detektierten Fälle einer invasiven Pilzinfektion wiesen die Lunge als primären Infektionsort auf. Der Großteil der Infektionen ereignete sich in den ersten 40 Tagen nach der Konditionierung, so waren 78% der Fälle als frühe Infektionen einzustufen. Als Risikofaktoren für die Entwicklung einer invasiven Mykose wurden in der vorliegenden Arbeit das Alter der Patienten und eine mit HLA-Missmatch durchgeführte Transplantation aufgedeckt. Die akute myeloische Leukämie als Grunderkrankung war ein weiterer, unabhängiger, prädisponierender Faktor für eine mögliche oder wahrscheinliche Mykose. Eine prolongierte aplastische Phase mit neutrophilen Granulozyten unter 100/µl sowie ein persistierendes neutropenes Fieber zeigten sich als Risikofaktoren für die Entwicklung einer möglichen Aspergillose. In Bezug auf wahrscheinliche Pilzinfektionen zeigte sich hier jedoch kein Zusammenhang. Eine lange Fieberphase präsentierte sich als Hinweisfaktor für eine folgende mögliche oder wahrscheinliche Infektion. Eine prolongierte Steroidzufuhr von mindestens 21 Tagen Dauer erwies sich als weiterer Risikofaktor für invasive mögliche Mykosen. Das Geschlecht der Patienten, akute sowie chronische Abstoßungsreaktionen, der CMV-Status von Empfänger und Spender und eine CMV-Reaktivierung, der HCT-CI Score, ein Nikotinabusus sowie respiratorische virale Infekte waren in der vorliegenden Studie keine signifikanten Risikofaktoren für eine invasive Pilzinfektion. Desweiteren waren im Studienkollektiv auch die Anzahl der verabreichten Antibiotika und ß-Lactam Antibiotika sowie die Dauer der neutropenen Phase mit weniger als 500 neutrophilen Granulozyten/µl keine relevanten Risikofaktoren für eine Pilzinfektion.
Es zeigte sich, dass Patienten, die an einer invasiven Mykose erkrankt waren, eine signifikant längere Krankenhausverweildauer hatten, als Patienten ohne Hinweis auf eine systemische Pilzinfektion. Nach einem halben Jahr Studienverlauf lag das Gesamtüberleben der Patienten bei 71,8% (ohne 2 unklare Fälle gerechnet). Jedoch war das Auftreten einer invasiven Pilzinfektion mit einer deutlich höheren Mortalität assoziiert. Nach 6 Monaten lebten von den Patienten mit einer wahrscheinlichen Mykose noch 33,3%, wohingegen von den Patienten, welche ohne Zeichen einer Mykose als unklassifiziert eingestuft wurden, noch 84% am Leben waren. Der Tod ereignete sich bei den Patienten mit einer möglichen oder wahrscheinlichen Infektion im Durchschnitt 40 Tage nach der Erstdiagnose der Pilzinfektion.
Zusammenfassend zeigte sich, dass der Anteil von Fällen mit invasiver Mykose im Studienkollektiv hoch lag und diese Infektion mit einer hohen Letalität assoziiert war. Anhand dieser Daten erscheint es indiziert, dass der Fokus bei allogen transplantierten Risiko-Patienten in Zukunft noch stärker auf dem Erkennen dieser opportunistischen Infektionen liegen sollte, um frühzeitig und adäquat intervenieren und therapieren zu können. Auch eine Aspergillus-aktive, antimykotische Prophylaxe ist für diesen Zeitraum zu prüfen. Da sich die ersten 40 Tage nach der Transplantation als Hauptrisiko-Zeitraum für eine invasive Mykose herausgestellt haben, sollten gerade in dieser, zum Großteil stationären, Phase besondere Anstrengungen unternommen werden, die erkrankten Patienten früh zu erkennen und zu behandeln. Auf diesem Wege wäre es eventuell möglich, das Gesamtüberleben der Patienten nach einer allogenen Stammzelltransplantation noch weiter zu verbessern.