Publications by authors named "Johannes C Fischer"

51 Publications

Association of HLA genotypes, AB0 blood type and chemokine receptor 5 mutant CD195 with the clinical course of COVID-19.

Eur J Med Res 2021 Sep 16;26(1):107. Epub 2021 Sep 16.

Medical Faculty, University of Dusseldorf, Dusseldorf, Germany.

Background: COVID-19, the pandemic disease caused by infection with SARS-CoV-2, may take highly variable clinical courses, ranging from symptom-free and pauci-symptomatic to fatal disease. The goal of the current study was to assess the association of COVID-19 clinical courses controlled by patients' adaptive immune responses without progression to severe disease with patients' Human Leukocyte Antigen (HLA) genetics, AB0 blood group antigens, and the presence or absence of near-loss-of-function delta 32 deletion mutant of the C-C chemokine receptor type 5 (CCR5).

Patient And Methods: An exploratory observational study including 157 adult COVID-19 convalescent patients was performed with a median follow-up of 250 days. The impact of different HLA genotypes, AB0 blood group antigens, and the CCR5 mutant CD195 were investigated for their role in the clinical course of COVID-19. In addition, this study addressed levels of severity and morbidity of COVID-19. The association of the immunogenetic background parameters were further related to patients' humoral antiviral immune response patterns by longitudinal observation.

Results: Univariate HLA analyses identified putatively protective HLA alleles (HLA class II DRB1*01:01 and HLA class I B*35:01, with a trend for DRB1*03:01). They were associated with reduced durations of disease instead decreased (rather than increased) total anti-S IgG levels. They had a higher virus neutralizing capacity compared to non-carriers. Conversely, analyses also identified HLA alleles (HLA class II DQB1*03:02 und HLA class I B*15:01) not associated with such benefit in the patient cohort of this study. Hierarchical testing by Cox regression analyses confirmed the significance of the protective effect of the HLA alleles identified (when assessed in composite) in terms of disease duration, whereas AB0 blood group antigen heterozygosity was found to be significantly associated with disease severity (rather than duration) in our cohort. A suggestive association of a heterozygous CCR5 delta 32 mutation status with prolonged disease duration was implied by univariate analyses but could not be confirmed by hierarchical multivariate testing.

Conclusion: The current study shows that the presence of HLA class II DRB1*01:01 and HLA class I B*35:01 is of even stronger association with reduced disease duration in mild and moderate COVID-19 than age or any other potential risk factor assessed. Prospective studies in larger patient populations also including novel SARS-CoV-2 variants will be required to assess the impact of HLA genetics on the capacity of mounting protective vaccination responses in the future.
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http://dx.doi.org/10.1186/s40001-021-00560-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8444184PMC
September 2021

Delayed skin reaction after mRNA-1273 vaccine against SARS-CoV-2: a rare clinical reaction.

Eur J Med Res 2021 Aug 25;26(1):98. Epub 2021 Aug 25.

Department of Radiation Oncology, University Hospital Medical Faculty, Heinrich-Heine-University, Moorenstr. 5, 40225, Düsseldorf, Germany.

Background: The coronavirus disease 2019 (COVID-19) is associated with a wide clinical spectrum of skin manifestations, including urticarial, vesicular, vasculitic and chilblain-like lesions. Recently, delayed skin reactions have been reported in 1% individuals following mRNA vaccination against SARS-CoV-2. The exact pathophysiology and the risk factors still remain unclear.

Patients And Methods: 6821 employees and patients were vaccinated at our institutions between February and June 2021. Every patient received two doses of the mRNA-1273 vaccine in our hospitals, and reported back in case of any side effects which were collected in our hospital managed database.

Results: Eleven of 6821 vaccinated patients (0.16%) developed delayed skin reactions after either the first or second dose of the mRNA-1273 vaccine against SARS-CoV-2. Eight of 11 patients (73%) developed a rash after the first dose, while in 3/11 (27%), the rash occurred after the second dose. More females (9/11) were affected. Four of 11 patients required antihistamines, with two needing additional topical steroids. All the cutaneous manifestations resolved within 14 days. None of the skin reactions after the first dose of the vaccine prevented the administration of the second dose. There were no long-term cutaneous sequelae in any of the affected individuals.

Conclusion: Our data suggests that skin reactions after the use of mRNA-1273 vaccine against SARS-CoV-2 are possible, but rare. Further studies need to be done to understand the pathophysiology of these lesions.
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http://dx.doi.org/10.1186/s40001-021-00557-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386154PMC
August 2021

Informed consent and informed intervention: SARS-CoV-2 vaccinations not just call for disclosure of newly emerging safety data but also for hypothesis generation and testing.

Eur J Med Res 2021 Aug 6;26(1):87. Epub 2021 Aug 6.

Department of Radiation Oncology, Medical Faculty, University Hospital Dusseldorf, Heinrich-Heine-University Dusseldorf, Moorenstr. 5, 40225, Düsseldorf, Germany.

Background: COVID-19 infection is a major threat to patients and health care providers around the world. One solution is the vaccination against SARS-CoV-2.

Methods: We performed a comprehensive query of the latest publications on the prevention of viral infections including the recent vaccination program and its side effects.

Results: The situation is evolving rapidly and there is no reasonable alternative to population-scale vaccination programs as currently enrolled.

Conclusion: Therefore, regulatory authorities should consider supplementing their conventional mandate of post-approval pharmacovigilance, which is based on the collection, assessment, and regulatory response to emerging safety findings.
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http://dx.doi.org/10.1186/s40001-021-00558-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8343366PMC
August 2021

Case Report: Convalescent Plasma Achieves SARS-CoV-2 Viral Clearance in a Patient With Persistently High Viral Replication Over 8 Weeks Due to Severe Combined Immunodeficiency (SCID) and Graft Failure.

Front Immunol 2021;12:645989. Epub 2021 May 3.

Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Duesseldorf, Medical Faculty, Heinrich-Heine-University Duesseldorf, Duesseldorf, Germany.

We describe the unique disease course and cure of SARS-CoV-2 infection in a patient with SCID and graft failure. In absence of a humoral immune response, viral clearance was only achieved after transfusion of convalescent plasma. This observation underscores the necessity of the humoral immune response for SARS-CoV-2 clearance.
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http://dx.doi.org/10.3389/fimmu.2021.645989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126709PMC
June 2021

Reconvalescent plasma/camostat mesylate in early SARS-CoV-2 Q-PCR positive high-risk individuals (RES-Q-HR): a structured summary of a study protocol for a randomized controlled trial.

Trials 2021 May 17;22(1):343. Epub 2021 May 17.

Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Duesseldorf, Medical Faculty Heinrich-Heine-University Duesseldorf, Moorenstr. 5, D-40225, Duesseldorf, Germany.

Objectives: Currently, there are no approved treatments for early disease stages of COVID-19 and few strategies to prevent disease progression after infection with SARS-CoV-2. The objective of this study is to evaluate the safety and efficacy of convalescent plasma (CP) or camostat mesylate administered within 72 h of diagnosis of SARS-CoV-2 infection in adult individuals with pre-existing risk factors at higher risk of getting seriously ill with COVID-19. Camostat mesylate acts as an inhibitor of the host cell serine protease TMPRSS2 and prevents the virus from entering the cell. CP represents another antiviral strategy in terms of passive immunization. The working hypothesis to be tested in the RES-Q-HR study is that the early use of CP or camostat mesylate reduces the likelihood of disease progression to (modified) WHO stages 4b-8 in SARS-CoV-2-positive adult patients at high risk of moderate or severe COVID-19 progression.

Trial Design: This study is a 4-arm (parallel group), multicenter, randomized (2:2:1:1 ratio), partly double-blind, controlled trial to evaluate the safety and efficacy of convalescent plasma (CP) or camostat mesylate with control or placebo in adult patients diagnosed with SARS-CoV-2 infection and high risk for progression to moderate/severe COVID-19. Superiority of the intervention arms will be tested.

Participants: The trial is conducted at 10-15 tertiary care centers in Germany. Individuals aged 18 years or above with ability to provide written informed consent with SARS-CoV-2 infection, confirmed by PCR within 3 days or less before enrolment and the presence of at least one SARS-CoV-2 symptom (such as fever, cough, shortness of breath, sore throat, headache, fatigue, smell/and or taste disorder, diarrhea, abdominal symptoms, exanthema) and symptom duration of not more than 3 days. Further inclusion criteria comprise: Presence of at least one of the following criteria indicating increased risk for severe COVID-19: Age > 75 years Chronic obstructive pulmonary disease (COPD) and/or pulmonary fibrosis BMI > 40 kg/m Age > 65 years with at least one other risk factor (BMI > 35 kg/m, coronary artery disease (CAD), chronic kidney disease (CKD) with GFR < 60 ml/min but ≥ 30 ml/min, diabetes mellitus, active tumor disease) BMI > 35 kg/m with at least one other risk factor (CAD, CKD with GFR < 60 ml/min but ≥ 30 ml/min, diabetes mellitus, active tumor disease) Exclusion criteria: 1. Age < 18 years 2. Unable to give informed consent 3. Pregnant women or breastfeeding mothers 4. Previous transfusion reaction or other contraindication to a plasma transfusion 5. Known hypersensitivity to camostat mesylate and/or severe pancreatitis 6. Volume stress due to CP administration would be intolerable 7. Known IgA deficiency 8. Life expectancy < 6 months 9. Duration SARS-CoV-2 typical symptoms > 3 days 10. SARS-CoV-2 PCR detection older than 3 days 11. SARS-CoV-2 associated clinical condition ≥ WHO stage 3 (patients hospitalized for other reasons than COVID-19 may be included if they fulfill all inclusion and none of the exclusion criteria) 12. Previously or currently hospitalized due to SARS-CoV-2 13. Previous antiviral therapy for SARS-CoV-2 14. ALT or AST > 5 x ULN at screening 15. Liver cirrhosis > Child A (patients with Child B/C cirrhosis are excluded from the trial) 16. Chronic kidney disease with GFR < 30 ml/min 17. Concurrent or planned anticancer treatment during trial period 18. Accommodation in an institution due to legal orders (§40(4) AMG). 19. Any psycho-social condition hampering compliance with the study protocol. 20. Evidence of current drug or alcohol abuse 21. Use of other investigational treatment within 5 half-lives of enrolment is prohibited 22. Previous use of convalescent plasma for COVID-19 23. Concomitant proven influenza A infection 24. Patients with organ or bone marrow transplant in the three months prior to screening visit INTERVENTION AND COMPARATOR: Participants will be randomized to the following 4 groups: 1) Convalescent plasma (CP), 2 units at screening/baseline visit (day 0) or day 1; CP is defined by the presence of neutralizing anti-SARS-CoV-2 antibodies with titers ≥ 1:160; individuals with body weight ≥ 150 kg will receive a third unit of plasma on day 3 2) Camostat mesylate (200 mg per capsule, one capsule taken each in the morning, afternoon and evening on days 1-7) 3) Standard of care (SOC, control for CP) 4) Placebo (identical in appearance to camostat mesylate capsules, one capsule taken each morning, afternoon and evening on days 1-7; for camostat mesylate control group) Participants will be monitored after screening/baseline on day 3, day 5, day 8, and day 14. On day 28 and day 56, telephone visits and on day 90, another outpatient visit are scheduled. Adverse events and serious adverse events will be monitored and reported until the end of the study. An independent data safety monitoring committee will review trial progression and safety.

Main Outcomes: The primary endpoint of the study is the cumulative number of individuals who progress to or beyond category 4b on the modified WHO COVID-19 ordinal scale (defined as hospitalization with COVID-19 pneumonia and additional oxygen demand via nasal cannula or mask) within 28 days after randomization.

Randomization: Participants will be randomized using the Alea-Tool ( aleaclinical.com ) in a 2:2:1:1 ratio to the treatment arms (1) CP, (2) camostat mesylate, (3) standard of care (SoC), and (4) placebo matching camostat mesylate. Randomization will be stratified by study center.

Blinding (masking): The camostat mesylate treatment arm and the respective placebo will be blinded for participants, caregivers, and those assessing outcomes. The treatment arms convalescent plasma and standard of care will not be blinded and thus are open-labeled, unblinded.

Numbers To Be Randomized (sample Size): Overall, n = 994 participants will be randomized to the following groups: n = 331 to convalescent plasma (CP), n = 331 to camostat mesylate, n = 166 to standard of care (SoC), and n = 166 to placebo matching camostat mesylate.

Trial Status: The RES-Q-HR protocol (V04F) was approved on the 18 December 2020 by the local ethics committee and by the regulatory institutions PEI/BfARM on the 2 December 2020. The trial was opened for recruitment on 26 December 2020; the first patient was enrolled on 7 January 2021 and randomized on 8 January 2021. Recruitment shall be completed by June 2021. The current protocol version RES-Q HR V05F is from 4 January 2021, which was approved on the 18 January 2021.

Trial Registration: EudraCT Number 2020-004695-18 . Registered on September 29, 2020. ClinicalTrial.gov NCT04681430 . Registered on December 23, 2020, prior to the start of the enrollment (which was opened on December 26, 2020).

Full Protocol: The full protocol (V05F) is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).
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http://dx.doi.org/10.1186/s13063-021-05181-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127198PMC
May 2021

Aging-associated distinctive DNA methylation changes of LINE-1 retrotransposons in pure cell-free DNA from human blood.

Sci Rep 2020 12 17;10(1):22127. Epub 2020 Dec 17.

Epigenetics Core Laboratory, Institute of Transplantation Diagnostics and Cell Therapeutics, Medical Faculty, Heinrich-Heine University Duesseldorf, Moorenstr. 5, 40225, Duesseldorf, Germany.

LINE-1 hypomethylation of cell-free DNA has been described as an epigenetic biomarker of human aging. However, in the past, insufficient differentiation between cellular and cell-free DNA may have confounded analyses of genome-wide methylation levels in aging cells. Here we present a new methodological strategy to properly and unambiguously extract DNA methylation patterns of repetitive, as well as single genetic loci from pure cell-free DNA from peripheral blood. Since this nucleic acid fraction originates mainly in apoptotic, senescent and cancerous cells, this approach allows efficient analysis of aged and cancerous cell-specific DNA methylation patterns for diagnostic and prognostic purposes. Using this methodology, we observe a significant age-associated erosion of LINE-1 methylation in cfDNA suggesting that the threshold of hypomethylation sufficient for relevant LINE-1 activation and consequential harmful retrotransposition might be reached at higher age. We speculate that this process might contribute to making aging the main risk factor for many cancers.
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http://dx.doi.org/10.1038/s41598-020-79126-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746734PMC
December 2020

Loss of Anti-SARS-CoV-2 Antibodies in Mild Covid-19.

N Engl J Med 2020 10 23;383(17):1694-1695. Epub 2020 Sep 23.

Heinrich Heine University, Düsseldorf, Germany

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http://dx.doi.org/10.1056/NEJMc2027051DOI Listing
October 2020

Measures of infection prevention and incidence of SARS-CoV-2 infections in cancer patients undergoing radiotherapy in Germany, Austria and Switzerland.

Strahlenther Onkol 2020 12 10;196(12):1068-1079. Epub 2020 Sep 10.

Medical Faculty, Department of Radiation Oncology, Heinrich Heine University, Duesseldorf, Germany.

Purpose: COVID-19 infection has manifested as a major threat to both patients and healthcare providers around the world. Radiation oncology institutions (ROI) deliver a major component of cancer treatment, with protocols that might span over several weeks, with the result of increasing susceptibility to COVID-19 infection and presenting with a more severe clinical course when compared with the general population. The aim of this manuscript is to investigate the impact of ROI protocols and performance on daily practice in the high-risk cancer patients during this pandemic.

Methods: We addressed the incidence of positive COVID-19 cases in both patients and health care workers (HCW), in addition to the protective measures adopted in ROIs in Germany, Austria and Switzerland using a specific questionnaire.

Results: The results of the questionnaire showed that a noteworthy number of ROIs were able to complete treatment in SARS-CoV‑2 positive cancer patients, with only a short interruption. The ROIs reported a significant decrease in patient volume that was not impacted by the circumambient disease incidence, the type of ROI or the occurrence of positive cases. Of the ROIs 16.5% also reported infected HCWs. About half of the ROIs (50.5%) adopted a screening program for patients whereas only 23.3% also screened their HCWs. The range of protective measures included the creation of working groups, instituting home office work and protection with face masks. Regarding the therapeutic options offered, curative procedures were performed with either unchanged or moderately decreased schedules, whereas palliative or benign radiotherapy procedures were more often shortened. Most ROIs postponed or cancelled radiation treatment for benign indications (88.1%). The occurrence of SARS-CoV‑2 infections did not affect the treatment options for curative procedures. Non-university-based ROIs seemed to be more willing to change their treatment options for curative and palliative cases than university-based ROIs.

Conclusion: Most ROIs reported a deep impact of SARS-CoV‑2 infections on their work routine. Modification and prioritization of treatment regimens and the application of protective measures preserved a well-functioning radiation oncology service and patient care.
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http://dx.doi.org/10.1007/s00066-020-01681-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483062PMC
December 2020

Face masks: benefits and risks during the COVID-19 crisis.

Eur J Med Res 2020 Aug 12;25(1):32. Epub 2020 Aug 12.

Department of Radiation Oncology, Heinrich Heine University, Dusseldorf, Germany.

Background: The German government has made it mandatory to wear respiratory masks covering mouth and nose (MNC) as an effective strategy to fight SARS-CoV-2 infections. In many countries, this directive has been extended on shopping malls or public transportation. The aim of this paper is to critically analyze the statutory regulation to wear protective masks during the COVID-19 crisis from a medical standpoint.

Methods: We performed an extensive query of the most recent publications addressing the prevention of viral infections including the use of face masks in the community as a method to prevent the spread of the infection. We addressed the issues of practicability, professional use, and acceptability based on the community and the environment where the user resided.

Results: Upon our critical review of the available literature, we found only weak evidence for wearing a face mask as an efficient hygienic tool to prevent the spread of a viral infection. However, the use of MNC seems to be linked to relevant protection during close contact scenarios by limiting pathogen-containing aerosol and liquid droplet dissemination. Importantly, we found evidence for significant respiratory compromise in patients with severe obstructive pulmonary disease, secondary to the development of hypercapnia. This could also happen in patients with lung infections, with or without SARS-CoV-2.

Conclusion: Epidemiologists currently emphasize that wearing MNC will effectively interrupt airborne infections in the community. The government and the politicians have followed these recommendations and used them to both advise and, in some cases, mandate the general population to wear MNC in public locations. Overall, the results seem to suggest that there are some clinically relevant scenarios where the use of MNC necessitates more defined recommendations. Our critical evaluation of the literature both highlights the protective effects of certain types of face masks in defined risk groups, and emphasizes their potential risks.
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http://dx.doi.org/10.1186/s40001-020-00430-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422455PMC
August 2020

The history and value of face masks.

Eur J Med Res 2020 Jun 23;25(1):23. Epub 2020 Jun 23.

Department of Radiation Oncology, Heinrich-Heine-University, Duesseldorf, Germany.

In the human population, social contacts are a key for transmission of bacteria and viruses. The use of face masks seems to be critical to prevent the transmission of SARS-CoV-2 for the period, in which therapeutic interventions are lacking. In this review, we describe the history of masks from the middle age to modern times.
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http://dx.doi.org/10.1186/s40001-020-00423-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309199PMC
June 2020

The role of passive immunization in the age of SARS-CoV-2: an update.

Eur J Med Res 2020 May 13;25(1):16. Epub 2020 May 13.

Department of Radiation Oncology, Heinrich Heine University, Moorenstr. 5, 40225, Düsseldorf, Germany.

The rapid spread of the corona virus pandemic is an existential problem for many people in numerous countries. So far, there is no effective vaccine protection or proven therapy available against the SARS-CoV-2 virus. In this review, we describe the role of passive immunization in times of the corona virus. Passive immunization could be a bridging technology to improve the immune defense of critically ill patients until better approaches with effective medications are available.
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http://dx.doi.org/10.1186/s40001-020-00414-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220618PMC
May 2020

Diagnostic Leukapheresis Enables Reliable Transcriptomic Profiling of Single Circulating Tumor Cells to Characterize Inter-Cellular Heterogeneity in Terms of Endocrine Resistance.

Cancers (Basel) 2019 Jun 28;11(7). Epub 2019 Jun 28.

Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, 40225 Duesseldorf, Germany.

Circulating tumor cells (CTCs) hold great promise with regard to prognosis, treatment optimization, and monitoring of breast cancer patients. Single CTC transcriptome profiling might help reveal valuable information concerning intra-patient heterogeneity relevant to therapeutic interventions. In this study, we combined Diagnostic Leukapheresis (DLA), which is a microfluidic enrichment using the Parsortix system, micromanipulation with CellCelector and subsequent single cell multi-marker transcriptome profiling. First, a PCR panel consisting of 30 different endocrine resistance and phenotypic marker genes was validated for single cell profiling by using different breast cancer cell lines. Second, this panel was applied to characterize uncultured and cultured CTCs, which were enriched from a cryopreserved DLA product obtained from a patient suffering from metastatic breast cancer resistant to endocrine therapy. Gene expression profiles of both CTC populations uncovered inter CTC heterogeneity for transcripts, which are associated with response or resistance to endocrine therapy (e.g., ). Hierarchical clustering revealed CTC subpopulations with different expressions of transcripts regarding the CTCs' differential phenotypes () and of transcripts involved in endocrine signaling pathways (). Moreover, ER-positive CTCs exhibited significant higher expression of Cyclin D1, which might be relevant for CDK4/6 inhibitor therapies. Overall, gene expression profiles of uncultured and cultured CTCs resulted in a partly combined grouping. Our findings demonstrate that multi-marker RNA profiling of enriched single uncultured CTCs and cultured CTCs form cryopreserved DLA samples may provide important insights into intra-patient heterogeneity relevant for targeted therapies and therapy resistance.
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http://dx.doi.org/10.3390/cancers11070903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679140PMC
June 2019

From in vitro to ex vivo: subcellular localization and uptake of graphene quantum dots into solid tumors.

Nanotechnology 2019 Sep 26;30(39):395101. Epub 2019 Jun 26.

Condensed Matter Physics Laboratory, Heinrich-Heine-University, D-40204 Düsseldorf, Germany.

Among various nanoparticles tested for pharmacological applications over the recent years, graphene quantum dots (GQDs) seem to be promising candidates for the construction of drug delivery systems due to their superior biophysical and biochemical properties. The subcellular fate of incorporated nanomaterial is decisive for transporting pharmaceuticals into target cells. Therefore a detailed characterization of the uptake of GQDs into different breast cancer models was performed. The demonstrated accumulation inside the endolysosomal system might be the reason for the particles' low toxicity, but has to be overcome for cytosolic or nuclear drug delivery. Furthermore, the penetration of GQDs into precision-cut mammary tumor slices was studied. These constitute a far closer to reality model system than monoclonal cell lines. The constant uptake into the depth of the tissue slices underlines the systems' potential for drug delivery into solid tumors.
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http://dx.doi.org/10.1088/1361-6528/ab2cb4DOI Listing
September 2019

Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products.

Clin Chem 2019 04 8;65(4):549-558. Epub 2019 Feb 8.

Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich Heine University Düsseldorf, Düsseldorf, Germany;

Introduction: Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients.

Methods: DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture.

Results: Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture.

Conclusions: The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.
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http://dx.doi.org/10.1373/clinchem.2018.296814DOI Listing
April 2019

Biomechanical Stability and Osteogenesis in a Tibial Bone Defect Treated by Autologous Ovine Cord Blood Cells-A Pilot Study.

Molecules 2019 Jan 15;24(2). Epub 2019 Jan 15.

Department of Orthopedics and Trauma Surgery, University of Duisburg-Essen, 45147 Essen, Germany.

The aim of this study was to elucidate the impact of autologous umbilical cord blood cells (USSC) on bone regeneration and biomechanical stability in an ovine tibial bone defect. Ovine USSC were harvested and characterized. After 12 months, full-size 2.0 cm mid-diaphyseal bone defects were created and stabilized by an external fixateur containing a rigidity measuring device. Defects were filled with (i) autologous USSC on hydroxyapatite (HA) scaffold (test group), (ii) HA scaffold without cells (HA group), or (iii) left empty (control group). Biomechanical measures, standardized X-rays, and systemic response controls were performed regularly. After six months, bone regeneration was evaluated histomorphometrically and labeled USSC were tracked. In all groups, the torsion distance decreased over time, and radiographies showed comparable bone regeneration. The area of newly formed bone was 82.5 ± 5.5% in the control compared to 59.2 ± 13.0% in the test and 48.6 ± 2.9% in the HA group. Labeled cells could be detected in lymph nodes, liver and pancreas without any signs of tumor formation. Although biomechanical stability was reached earliest in the test group with autologous USSC on HA scaffold, the density of newly formed bone was superior in the control group without any bovine HA.
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http://dx.doi.org/10.3390/molecules24020295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358876PMC
January 2019

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting.

Cytometry A 2018 12;93(12):1213-1219

Faculty of the Heinrich-Heine, General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty, University Düsseldorf, Düsseldorf, North Rhine-Westphalia, Germany.

Diagnostic leukapheresis (DLA) is based on continuous centrifugation that collects mononuclear cells from peripheral blood with a density of 1.055-1.08 g/ml. As epithelial cells have a similar density, DLA cocollects circulating tumor cell (CTCs) along with the targeted mononuclear cells. Here, we report on our single center experience applying DLA in 40 nonmetastatic and metastatic breast cancer patients and its impact on CTC detection. We found that the use of just 5% of the DLA product (corresponding to a median peripheral blood volume of around 60 ml) in the CellSearch® assay already leads to a significant increase in CTC detection frequency and yield. The implementation of the method was unproblematic, and we did not observe any adverse events in our patient cohort. Extrapolating the CTC counts in the DLA samples to the whole DLA product indicated that enormous CTC numbers could be harvested by this approach (around 205x more CTCs than in the 7.5 ml blood sample in M1 patients). In conclusion, DLA is a clinically safe method to collect CTCs from liters of blood enabling a real liquid biopsy. Yet, further technical developments are required to process whole DLA products and exploit the full potential of this approach. As it is foreseeable that DLA will be used by several groups, and hopefully ultimately brought to the patients in a routine setting, we discuss recommendations on the minimum of required information for reporting on DLAs to allow comparison across different approaches. © 2018 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23669DOI Listing
December 2018

Genome-wide hypomethylation of LINE-1 and Alu retroelements in cell-free DNA of blood is an epigenetic biomarker of human aging.

Saudi J Biol Sci 2018 Sep 13;25(6):1220-1226. Epub 2018 Feb 13.

Epigenetics Core Laboratory, Institute of Transplantation Diagnostics and Cell Therapeutics, Medical Faculty, Heinrich-Heine University Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany.

Aging associated DNA hypomethylation of LINE-1 and Alu retroelements may be a crucial determinant of loss of genomic integrity, deterioration and cancer. In peripheral blood LINE-1 hypomethylation has been reported to increase during aging, but other studies did not observe significant changes. We hypothesized that these apparently inconsistent reports might relate to differences between cellular and cell-free DNA. Using the technique of idiolocal normalization of real-time methylation-specific PCR (IDLN-MSP) for genetic imbalanced DNA specimens we obtained evidence that LINE-1 hypomethylation in cell-free DNA, but not cellular DNA from peripheral blood is an epigenetic biomarker for human aging. Furthermore, hypomethylation of cell-free DNA is more extensive in smokers, suggesting that it might be used as a surrogate marker for monitoring the improvement of smoking-induced adverse effects after cancelling smoking.
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http://dx.doi.org/10.1016/j.sjbs.2018.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117241PMC
September 2018

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Int J Cancer 2018 11 19;143(10):2584-2591. Epub 2018 Sep 19.

Prostate Cancer Targeted Therapies Group, The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, Sutton, UK.

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.
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http://dx.doi.org/10.1002/ijc.31752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637919PMC
November 2018

Isolation of circulating tumor cells from pancreatic cancer by automated filtration.

Oncotarget 2017 Oct 16;8(49):86143-86156. Epub 2017 Sep 16.

Bayer AG, Biomarker Research, 13353 Berlin, Germany.

It is now widely recognized that the isolation of circulating tumor cells based on cell surface markers might be hindered by variability in their protein expression. Especially in pancreatic cancer, isolation based only on EpCAM expression has produced very diverse results. Methods that are independent of surface markers and therefore independent of phenotypical changes in the circulating cells might increase CTC recovery also in pancreatic cancer. We compared an EpCAM-dependent (IsoFlux) and a size-dependent (automated Siemens Healthineers filtration device) isolation method for the enrichment of pancreatic cancer CTCs. The recovery rate of the filtration based approach is dramatically superior to the EpCAM-dependent approach especially for cells with low EpCAM-expression (filtration: 52%, EpCAM-dependent: 1%). As storage and shipment of clinical samples is important for centralized analyses, we also evaluated the use of frozen diagnostic leukapheresis (DLA) as source for isolating CTCs and subsequent genetic analysis such as mutation detection analysis. Using frozen DLA samples of pancreatic cancer patients we detected CTCs in 42% of the samples by automated filtration.
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http://dx.doi.org/10.18632/oncotarget.21026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689673PMC
October 2017

Disseminated tumour cells with highly aberrant genomes are linked to poor prognosis in operable oesophageal adenocarcinoma.

Br J Cancer 2017 Aug 20;117(5):725-733. Epub 2017 Jul 20.

Department of General, Visceral and Paediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine-University Duesseldorf, Moorenstr. 5, Duesseldorf 40225, Germany.

Background: Chromosomal instability (CIN) has repeatedly been identified as a prognostic marker. Here we evaluated the percentage of aberrant genome per cell (PAG) as a measure of CIN in single disseminated tumour cells (DTC) isolated from patients with operable oesophageal adenocarcinoma (EAC), to assess the impact of CIN DTCs on prognosis.

Methods: We isolated CK18 DTCs from bone marrow (BM) or lymph node (LN) preparations of operable EAC patients. After whole-genome amplification, single DTCs were analysed for chromosomal gains and losses using metaphase-based comparative genomic hybridisation (mCGH). We calculated the PAG for each DTC and determined the critical threshold value that identifies high-risk patients by STEPP (Subpopulation Treatment Effect Pattern Plot) analysis in two independent EAC patient cohorts (cohort #1, n=44; cohort #2; n=29).

Results: The most common chromosomal alterations observed among the DTCs were typical for EAC, but the DTCs showed a varying PAG between individual patients. Generally, LNDTCs displayed a significantly higher PAG than BMDTCs. STEPP analysis revealed an increasing PAG of DTCs to be correlated with an increased risk for short survival in two independent EAC cohorts as well as in the corresponding pooled analysis. In all three data sets (cohort #1, cohort #2 and pooled cohort), PAG DTCs conferred an independent risk for a significantly decreased survival.

Conclusions: The analysis of PAG/CIN in solitary marker-positive DTCs identifies operable EAC patients with poor prognosis, indicating a more aggressive minimal residual disease.
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http://dx.doi.org/10.1038/bjc.2017.233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572184PMC
August 2017

Recipient HLA-C Haplotypes and microRNA 148a/b Binding Sites Have No Impact on Allogeneic Hematopoietic Cell Transplantation Outcomes.

Biol Blood Marrow Transplant 2017 01 13;23(1):153-160. Epub 2016 Oct 13.

University of Colorado, Pediatric BMT and Cell Therapy, Aurora, Colorado. Electronic address:

Natural killer cells are important in graft-versus-leukemia responses after hematopoietic cell transplantation (HCT). A variety of surface receptors dictates natural killer cell function, including killer cell immunoglobulin-like receptor recognition of HLA-C. Previous single-center studies show that HLA-C epitopes, designated C1 and C2, were associated with allogeneic HCT outcomes; specifically, recipients homozygous for the C1 epitope (C1/C1) experienced a survival benefit. Additionally, mismatching at HLA-C was beneficial in recipients possessing at least 1 C2 allele, whereas the opposite was true for homozygous C1 (C1/C1) recipients where HLA-C mismatching resulted in worse outcomes. In this analysis we aimed to validate these findings in a large multicenter study. We also set out to determine whether surface expression of recipient HLA-C, determined by polymorphism in a microRNA (miR-148a/b) binding site within the 3'-region of the HLA-C transcript, was associated with transplant outcomes. In this large registry cohort, we were unable to confirm the prior findings regarding recipient HLA-C epitope status and outcome. Additionally, HLA-C surface expression (ie, surface density), as predicted by the miR-148a/b binding single nucleotide polymorphism, was also not with associated transplant outcomes. Collectively, neither HLA-C surface expression, as determined by miR-148a/b, nor recipient HLA-C epitopes (C1, C2) are associated with allogeneic HCT outcomes.
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http://dx.doi.org/10.1016/j.bbmt.2016.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198579PMC
January 2017

Human glioblastoma stem-like cells accumulate protoporphyrin IX when subjected to exogenous 5-aminolaevulinic acid, rendering them sensitive to photodynamic treatment.

J Photochem Photobiol B 2016 Oct 27;163:203-10. Epub 2016 Aug 27.

Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine University Hospital Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany. Electronic address:

Glioblastoma (GBM) is the most frequent and lethal primary brain tumor in adults. Despite multimodal therapy combining resection, radio- and alkylating chemotherapy, disease recurrence is universal and prognosis of patients is poor. Glioblastoma stem-like cells (GSC), which can be grown as neurospheres from primary tumors in vitro, appear to be resistant to the established therapies and are suspected to be the driving force for disease recurrence. Thus, efficacy of emerging therapies may depend on targeting GSC. 5-aminolaevulinic acid-mediated photodynamic therapy (5-ALA/PDT) is a promising therapeutic approach in GBM. It utilizes the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in GBM cells after application of 5-ALA. When exposed to laser light of 635nm wavelength, PPIX initiates a photochemical reaction resulting in the generation of reactive oxygen species, which kill the tumor cells. Whether GSC accumulate PPIX and are sensitive to 5-ALA/PDT is currently unknown. Therefore, human GSC were derived from primary tumors and grown as neurospheres under serum free conditions. When subjected to exogenous 5-ALA, a dose- and time-dependent accumulation of PPIX in GSC was observed by flow cytometry, which varied between individual GSC preparations. Subsequent exposure to laser light of 635nm wavelength substantially killed GSC, whereas treatment with 5-ALA or exposure to laser light only had no effect. LD50 values differed between GSC preparations, but were negatively correlated with PPIX accumulation in GSC. In summary, we report for the first time that glioblastoma stem-like cells accumulate PPIX when subjected to 5-aminolaevulinic acid and are sensitive to 5-aminolaevulinc acid based photodynamic therapy.
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http://dx.doi.org/10.1016/j.jphotobiol.2016.08.043DOI Listing
October 2016

Effects of thromboprophylaxis on mesenchymal stromal cells during osteogenic differentiation: an in-vitro study comparing enoxaparin with rivaroxaban.

BMC Musculoskelet Disord 2016 Mar 1;17:108. Epub 2016 Mar 1.

Department of Orthopaedic Surgery, Heinrich-Heine-University, Moorenstr. 5, 40225, Düsseldorf, Germany.

Background: Low-molecular-weight heparins (e.g. Enoxaparin) are widely used to prevent venous thromboembolism after orthopaedic surgery, but there are reports about serious side effects including reduction in bone density and strength. In recent years new oral antithrombotic drugs (e.g. direct Factor Xa-inhibitor, Rivaroxaban) have been used to prevent venous thromboembolism. However, there is lack of information on the effects of these new drugs on human mesenchymal stromal cells during osteogenic differentiation and, therefore, effects during postoperative bone healing.

Methods: We evaluated the effects of Rivaroxaban and Enoxaparin on the proliferation, mRNA and surface receptor expression as well as differentiation capacity of primary human mesenchymal stromal cells during their osteogenic differentiation.

Results: Enoxaparin, but not Rivaroxaban treatment significantly increased human mesenchymal stromal cell (hMSC) proliferation during the first week of osteogenic differentiation while suppressing osteogenic marker genes, surface receptor expression and calcification.

Conclusions: This is the first paper to demonstrate that Rivaroxaban had no significant influence on hMSC differentiation towards the osteogenic lineage, indicating a less affected bone healing process compared with Enoxaparin in vitro. Based on these findings Rivaroxaban seems to be superior to Enoxaparin in early stages of bone healing in vitro.
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http://dx.doi.org/10.1186/s12891-016-0966-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772441PMC
March 2016

Challenges for CTC-based liquid biopsies: low CTC frequency and diagnostic leukapheresis as a potential solution.

Expert Rev Mol Diagn 2016 16;16(2):147-64. Epub 2015 Dec 16.

d Department of Medical Cell BioPhysics, MIRA Institute , University of Twente , Enschede , The Netherlands.

Circulating tumor cells (CTCs) are very attractive surrogate markers for systemic cancer. Currently, major efforts are being made to use these rare cells in the sense of a liquid biopsy to gain molecular information for rational therapeutic decision-making. The advancements in molecular analyses of CTCs down to the single-cell level have been significant in recent years and some applications are ready to be used in clinical studies. As discussed in this review, a major challenge for translating such molecular CTC-based assays into the clinic is the extremely low frequency of CTCs and the associated problems of their reliable detection and isolation. A potential solution to overcome the low CTC frequency is the recently introduced diagnostic leukapheresis that permits screening of liters of blood. Discussed here are the challenges as well as the current efforts implementing this method into clinical workflows to realize more reliable liquid biopsies.
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http://dx.doi.org/10.1586/14737159.2016.1123095DOI Listing
November 2016

Selective downregulation of HLA-C and HLA-E in childhood acute lymphoblastic leukaemia.

Br J Haematol 2016 08 3;174(3):477-80. Epub 2015 Nov 3.

Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University, Düsseldorf, Germany.

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http://dx.doi.org/10.1111/bjh.13777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854806PMC
August 2016

Influence of Di(2-ethylhexyl)phthalate on migration rate and differentiation of human hematopoietic stem and progenitor cells (CD34+).

Clin Hemorheol Microcirc 2015 ;61(2):111-8

Hochschule Furtwangen University, Villingen-Schwenningen, BW, Germany.

Introduction: Phthalates are a group of synthetic plasticizers that are ubiquitous environmental pollutants with toxic and endocrine disrupting characteristics. DEHP is the most commonly used plasticizer in the world and is still applied to stem cell transfusion bags used for storage of hematopoietic stem and progenitor cells (CD34+ HSPC), which are transferred during stem cell transplantation. Here we examined the effect of DEHP on vitality of CD34+ HSPC as well as stem cell specific properties like migration and differentiation capacity - both important for successful stem cell transplantations.

Material And Methods: CD34+ HSPC were incubated for 24 h and 72 h with DEHP concentrations ranging from 1 μg/ml to 250 μg/ml. DEHP was diluted in DMSO. Migration rate was analyzed along an SDF-1α gradient using Transwell migration inserts. Differentiation of CD34+ HSPC was investigated after two weeks in methylcellulose with colony stimulating factors. Apoptosis rate was measured via Annexin V and 7-AAD staining.

Results: 24 h of incubation with 10 μg/ml DEHP led to a significant (p <  0.01) decrease in migration rate of CD34+ HSPC (70.70% ± 7.53% ) with a minimum migration rate of 48.33% ± 6.72% in relation to control after incubation with 100 μg/ml DEHP for 72 h. Incubation with the highest tested DEHP concentrations (50 and 100 μg/ml) significantly (p <  0.05) altered colony formation rate and cell type distribution. Apoptosis rate of CD34+ HSPC significantly (p <  0.05) increased after incubation with concentrations of 10 μg/ml DEHP for 24 h (1.46 ± 0.19) with a maximum apoptosis rate of 2.71 ± 0.66 after 24 h incubation with the highest DEHP concentration (250 μg/ml) in relation to control.

Conclusions: As shown, DEHP takes impact on migration rate, apoptosis rate, and differentiation of CD34+ HSPC. As these are functions with an important role in stem cell transplantations, the usage of DEHP-free stem cell transfusion bags should be considered.
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http://dx.doi.org/10.3233/CH-151984DOI Listing
June 2016

Deep serum discoveries: SDF-1α and HSA fragments in myelodysplastic syndromes.

Am J Hematol 2015 Sep 27;90(9):E185-7. Epub 2015 Jul 27.

Department of Clinical Biochemistry and Pathobiochemistry, Leibniz Center for Diabetes Research, Heinrich-Heine-University, 40225, Düsseldorf, Germany.

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http://dx.doi.org/10.1002/ajh.24070DOI Listing
September 2015

Age-Related Increase of EED Expression in Early Hematopoietic Progenitor Cells is Associated with Global Increase of the Histone Modification H3K27me3.

Stem Cells Dev 2015 Sep 23;24(17):2018-31. Epub 2015 Jun 23.

2 Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Düsseldorf , Medical Faculty, Düsseldorf, Germany .

Human hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood exhibit higher differentiation potential and repopulation capacity compared to adult HSPCs. The molecular basis for these functional differences is currently unknown. Upon screening for epigenetic effector genes being differentially expressed in neonatal and adult HSPC subpopulations, the Polycomb Repressive Complex 2 (PRC2) member EED was identified. Even though EED is expressed at comparable amounts in neonatal and adult multipotent HSPCs, early adult lineage committed progenitors of the lymphomyeloid (LM) and erythromyeloid lineages expressed higher EED amounts than neonatal HPCs. We demonstrate that EED overexpression directly leads to higher H3K27me3 levels, a repressive histone modification that is mediated by the PRC2 complex. Quantitative analysis of H3K27me3 levels by FPLC-based ELISA revealed elevated levels in primary blood cells from adults. Besides quantitative changes, gene ontology analysis of the genome-wide H3K27me3 distribution revealed qualitative changes in adult HSPCs with elevated levels in genes associated with nonhematopoietic development pathways. In contrast, H3K4me3 which labels active chromatin was enriched on hematopoietic genes. In vitro differentiation of EED-transfected neonatal HSPCs revealed aberrant expression of the myelopoietic marker CD14, suggesting that EED affects the lymphoid versus myeloid decision processes within the lymphomyeloid lineage. This is in line with LM progenitors having the most pronounced differences in EED expression. Highlighting the dynamic roles of epigenetic modifications in human hematopoiesis, the present data demonstrate shifts in the PRC2-associated histone modification H3K27me3 from birth to adulthood.
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http://dx.doi.org/10.1089/scd.2014.0435DOI Listing
September 2015
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