Regulation of blood-screening in vitro diagnostics in sub-Saharan African countries remains a challenge.

According to the World Health Organization, blood must be screened for major transmitted infections before transfusion to prevent the possibility of passing an infection to the recipient. For accurate detection of infectious disease pathogens in the blood of donors, in-vitro diagnostic medical devices (IVDs) of high specificity and sensitivity should be used. In mature healthcare systems, the regulatory authorities authorize the usage of devices with the highest performance capabilities, which are also controlled through active market oversight. However, in Sub-Saharan African countries, the regulation of IVDs is often poorly developed. With the lack of stringent regulatory oversight, IVDs of poor quality can be put on the market and used for blood donor screening, which, ultimately, poses a great public health threat. The BloodTrain is a humanitarian project from the Germany Federal Ministry of Health that aims to help strengthen the regulatory authorities in Sub-Saharan partner countries. Here, we present the status of IVD regulation in the partner countries and the objectives that the BloodTrain project aims to achieve in the region toward regulating IVDs.

Strengthening Blood Regulatory Systems to Tackle Africa's Unmet Needs for Blood and Blood Products.

Background: Assuring the quality and safety of blood and blood components is an essential element of health care in all countries and requires government commitment and legal frameworks. Ineffective regulation of blood and blood components has far-reaching consequences that are not limited to the affected countries but also have extensive global implications. Summary: In this review, we summarize the work of the project BloodTrain funded by the German Ministry of Health within the framework of the Global Health Protection Programme to strengthen regulatory structures in Africa that are imperative to guarantee the improved availability, safety, and quality of blood and blood products. Key Messages: Intense interaction with the stakeholders in African partner countries lead to first measurable successes in the strengthening of blood regulation, as shown here for hemovigilance.

Report on the GHPP-BloodTrain online workshop on "Authorisation and Licensing of Blood Establishments - Review of quality documentation of blood and blood components", 5th-8th of July 2021.

The GHPP BloodTrain team developed an e-learning concept in response to the COVID-19 travel restrictions, providing training formats with virtual stages that can be completed during the pandemic (and beyond) and on-site stages, where practical exercises and case reports in smaller groups can lead to a deeper understanding of the content. The virtual training workshop on "Authorisation and Licensing of Blood Establishments", hosted by the PEI GHPP BloodTrain from the 5th to the 8th of July 2021, was the first application of this concept. The number of participants could be substantially increased compared to an on-site event thanks to the virtual setting. Participants came mainly from national regulatory authorities and national blood transfusion services. There were also some Ministry of Health representatives from 19 countries from the WHO regions of AFRO, EMRO, and from Indonesia in attendance. The virtual workshop focused on reviewing and evaluating the quality documentation required for approval of processes used by blood establishments to prepare blood components. Presentations were given by members of the GHPP BloodTrain team as well as by representatives of the German Red Cross. The program was complemented by contributions from the WHO HQ and presentations on country-specific experiences from Ghana and Zimbabwe.

Congress report: Online workshop on assessment of technical files for blood screening in vitro diagnostics for sub-Sahara African countries.

OBJECTIVES: The online workshop on IVD regulation was performed to broaden the understanding of the technical documentation needed for IVD licensing and the strategies to asses it. BACKGROUND: Testing of blood donors and donations significantly reduces the risk of transmitting transfusion-transmissible infections. Many test systems are commercially available, but not all meet the recommended sensitivity and specificity standards. Many African countries either lack functional structures for the regulation of IVDs this poses a threat to the quality of the blood supply. MATERIALSAND METHODS: The Paul-Ehrlich-Institut BloodTrain organised an online workshop in September 2021 to introduce staff from several National Regulatory Authorities (NRAs) in Africa to the regulation of IVD and the technical information that need to be provided by the manufacturers of blood screening IVD. Their evaluation was trained in practical exercises. RESULTS: This online workshop brought together over hundred participants from NRAs of 12 African countries. Speakers from PEI, Blood Train, WHO and academia, with experience in IVD regulation trained participants in the various topics addressed during this workshop. CONCLUSIONS: This workshop presented a great starting point for most participating NRAs to set up and/or strengthen their regulatory structures for IVDs.

In vitro diagnostics for screening the blood supply: the new European regulation for IVD and the WHO IVD prequalification programme.

Blood transfusion remains a routine life-saving medical procedure that helps replace blood lost due to surgery, injury or disease. The quality of transfused blood is crucial in this process as blood donors must be free of transfusion-transmissible infections and donated blood should be compatible to that of the recipient. The quality of donated blood could be affected by the quality of in vitro diagnostic medical devices (IVDs) used in the screening process. Consequently, the need for high-quality, safe and well-performing IVDs for use in transfusion medicine arises, accompanied by the need for tight regulations in this domain. In the European Union, the new IVD Regulation will replace the existing IVD Directive within a five-year transitional period. Manufacturers of IVDs are expected to fully comply with the new Regulation by 26 May 2022. In this review, we address the major differences relating to marketing authorization and testing between this new Regulation and its predecessor. We further present the main elements of the prequalification assessment introduced by the WHO for IVDs, including disease-specific IVDs for blood screening laboratories.

Report from the GHPP-BloodTrain inspection workshop in Harare the 20th to the 24th of May 2019.

During the training workshop on Inspection of Blood Establishments, which was hosted by the PEI GHPP BloodTrain in Harare from the 20th to the 24th of May 2019, participants from the National Regulatory Authorities from seven Sub-Sahara African countries presented their current experiences related to regulation and inspection of blood establishments in their respective countries. While in all seven countries regulation and inspection of conventional medicinal products manufacturer is performed, the regulatory situation of blood and blood components as well as inspection of blood establishments is still heterogeneous.

Preclinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies.

Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.

[Scientific advice by the national and European approval authorities concerning advanced therapy medicinal products]. / Wissenschaftliche Beratung durch nationale und europäische Zulassungsbehörden bei Arzneimitteln für neuartige Therapien.

The aim of scientific advice is to support pharmaceutical developers in regulatory and scientific questions, thus facilitating the development of safe and efficacious new medicinal products. Recent years have shown that the development of advanced therapy medicinal products (ATMPs) in particular needs a high degree of regulatory support. On one hand, this is related to the complexity and heterogeneity of this group of medicinal products and on the other hand due to the fact that mainly academic research institutions and small- and medium-sized enterprises (SMEs) are developing ATMPs. These often have limited regulatory experience and resources. In 2009 the Paul-Ehrlich-Institut (PEI) initiated the Innovation Office as a contact point for applicants developing ATMPs. The mandate of the Innovation Office is to provide support on regulatory questions and to coordinate national scientific advice meetings concerning ATMPs for every phase in drug development and especially with view to the preparation of clinical trial applications. On the European level, the Scientific Advice Working Party (SAWP) of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicinal Agency (EMA) offers scientific advice. This article describes the concepts of national and EMA scientific advice concerning ATMPs and summarizes the experience of the last six years.

Concise review: workshop review: understanding and assessing the risks of stem cell-based therapies.

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting-edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products.

European regulatory tools for advanced therapy medicinal products.

Increasing scientific knowledge and technical innovations in the areas of cell biology, biotechnology and medicine resulted in the development of promising therapeutic approaches for the prevention and treatment of human diseases. Advanced therapy medicinal products (ATMPs) reflect a complex and innovative class of biopharmaceuticals as these products are highly research-driven, characterised by innovative manufacturing processes and heterogeneous with regard to their origin, type and complexity. This class of ATMP integrates gene therapy medicinal products, somatic cell therapy medicinal products and tissue engineering products and are often individualized and patient-specific products. Multiple challenges arise from the nature of ATMPs, which are often developed by micro, small and medium sized enterprises, university and academia, for whom regulatory experiences are limited and regulatory requirements are challenging. Regulatory guidance such as the reflection paper on classification of ATMPs and guidelines highlighting product-specific issues support academic research groups and pharmaceutical companies to foster the development of safe and effective ATMPs. This review provides an overview on the European regulatory aspects of ATMPs and highlights specific regulatory tools such as the ATMP classification procedure, a discussion on the hospital exemption for selected ATMPs as well as borderline issues towards transplants/transfusion products.

Regulatory structures for gene therapy medicinal products in the European Union.

Taking into account the complexity and technical specificity of advanced therapy medicinal products: (gene and cell therapy medicinal products and tissue engineered products), a dedicated European regulatory framework was needed. Regulation (EC) No. 1394/2007, the "ATMP Regulation" provides tailored regulatory principles for the evaluation and authorization of these innovative medicines. The majority of gene or cell therapy product development is carried out by academia, hospitals, and small- and medium-sized enterprises (SMEs). Thus, acknowledging the particular needs of these types of sponsors, the legislation also provides incentives for product development tailored to them. The European Medicines Agency (EMA) and, in particular, its Committee for Advanced Therapies (CAT) provide a variety of opportunities for early interaction with developers of ATMPs to enable them to have early regulatory and scientific input. An important tool to promote innovation and the development of new medicinal products by micro-, small-, and medium-sized enterprises is the EMA's SME initiative launched in December 2005 to offer financial and administrative assistance to smaller companies. The European legislation also foresees the involvement of stakeholders, such as patient organizations, in the development of new medicines. Considering that gene therapy medicinal products are developed in many cases for treatment of rare diseases often of monogenic origin, the involvement of patient organizations, which focus on rare diseases and genetic and congenital disorders, is fruitful. Two such organizations are represented in the CAT. Research networks play another important role in the development of gene therapy medicinal products. The European Commission is funding such networks through the EU Sixth Framework Program.

PKCδ-induced PU.1 phosphorylation promotes hematopoietic stem cell differentiation to dendritic cells.

Human CD34(+) hematopoietic stem cells (HSCs) exhibit the potential to differentiate into a variety of specialized blood cells. The distinct intracellular mechanisms that control cell fate and lineage commitment of these multipotent cells are not well defined. In this study, we investigate and modulate the signaling processes during HSC differentiation toward myeloid dendritic cells (mDCs). DC differentiation induced by the cytokines Granulocyte macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4) led to activation of the Extracellular-signal-regulated kinase (ERK), protein kinase C (PKC), and Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) but not the SAPK/c-Jun NH(2) -terminal kinase and p38 mitogen-activated protein kinase signaling pathways. From the activated signaling pathways the PKC isoform δ was found to phosphorylate the transcription factor PU.1, which is described as one of the key factors for myeloid HSC differentiation. On molecular level, PKCδ regulated PU.1 activity by affecting its transactivation activity, whereas its DNA binding activity remained unaffected. This was accompanied by PKCδ-induced phosphorylation of the PU.1 transactivation domain. Furthermore, treatment with PKC- and ERK1/2-specific signaling inhibitors impaired both HSC differentiation toward mDCs as well as phosphorylation-mediated transactivation activity of PU.1. Taken together, these results provide new insights into the molecular mechanisms promoting the differentiation process of HSCs toward mDCs and introduce the PKC isoform δ as critical mediator.

Assessing the safety of stem cell therapeutics.

Unprecedented developments in stem cell research herald a new era of hope and expectation for novel therapies. However, they also present a major challenge for regulators since safety assessment criteria, designed for conventional agents, are largely inappropriate for cell-based therapies. This article aims to set out the safety issues pertaining to novel stem cell-derived treatments, to identify knowledge gaps that require further research, and to suggest a roadmap for developing safety assessment criteria. It is essential that regulators, pharmaceutical providers, and safety scientists work together to frame new safety guidelines, based on "acceptable risk," so that patients are adequately protected but the safety "bar" is not set so high that exciting new treatments are lost.

Endocardial radiofrequency ablation for hypertrophic obstructive cardiomyopathy: acute results and 6 months' follow-up in 19 patients.

OBJECTIVES: The purpose of this study was to examine the efficacy and safety of endocardial radiofrequency ablation of septal hypertrophy (ERASH) for left ventricular outflow tract (LVOT) gradient reduction in hypertrophic obstructive cardiomyopathy (HOCM). BACKGROUND: Anatomic variability of the vessels supplying the obstructing septal bulge can limit the efficacy of transcoronary ablation of septal hypertrophy in HOCM. Previous studies showed that inducing a local contraction disorder without reducing septal mass results in effective gradient reduction. We examined an alternative endocardial approach to transcoronary ablation of septal hypertrophy by using ERASH. METHODS: Nineteen patients with HOCM were enrolled; in 9 patients, the left ventricular septum was ablated, and in 10 patients, the right ventricular septum was ablated. Follow-up examinations (echocardiography, 6-min walk test, bicycle ergometry) were performed 3 days and 6 months after ERASH. RESULTS: After 31.2 ± 10 radiofrequency pulses, a significant and sustained LVOT gradient reduction could be achieved (62% reduction of resting gradients and 60% reduction of provoked gradients, p = 0.0001). The 6-min walking distance increased significantly from 412.9 ± 129 m to 471.2 ± 139 m after 6 months, p = 0.019); and New York Heart Association functional class was improved from 3.0 ± 0.0 to 1.6 ± 0.7 (p = 0.0001). Complete atrioventricular block requiring permanent pacemaker implantation occurred in 4 patients (21%); 1 patient had cardiac tamponade. CONCLUSIONS: ERASH is a new therapeutic option in the treatment of HOCM, allowing significant and sustained reduction of the LVOT gradient as well as symptomatic improvement with acceptable safety by inducing a discrete septal contraction disorder. It may be suitable for patients not amenable to transcoronary ablation of septal hypertrophy or myectomy.

Predictors of complete heart block after transcoronary ablation of septal hypertrophy: results of a prospective electrophysiological investigation in 172 patients with hypertrophic obstructive cardiomyopathy.

OBJECTIVES: This study analyzed changes in intracardiac conduction during transcoronary ablation of septal hypertrophy (TASH) to identify predictors for pacemaker dependency after TASH. BACKGROUND: Transcoronary ablation of septal hypertrophy is an accepted therapeutic option in hypertrophic obstructive cardiomyopathy (HOCM). However, atrioventricular conduction disorders, requiring permanent pacemaker implantation, remain a major adverse effect. METHODS: This study measured changes in intracardiac conduction in 172 consecutive patients during TASH by simultaneously recording electrophysiological parameters and correlated these parameters with the occurrence of complete heart block during continuous electrocardiographic monitoring for 8 days. RESULTS: Intraprocedural complete heart block occurred in 36 patients (20.1%) and was associated with a pre-existing bundle branch block (p = 0.010) or advanced age (p = 0.023). All patients with delayed complete heart block during follow-up (n = 15, 8.7%), occurring 1 to 6 days after TASH, showed lack of retrograde atrioventricular nodal conduction after TASH (p = 0.018). None of the patients with intact retrograde conduction after TASH developed delayed complete heart block. Further risk factors for delayed block were advanced age, intraprocedural complete heart block, and prolonged QRS duration before or after TASH (p < 0.05 for all). Permanent pacemaker implantation was performed in 20 patients. CONCLUSIONS: Measurement of intracardiac conduction during TASH improves the safety of the procedure by enabling identification of patients who are at risk of complete heart block after TASH. The duration of prophylactic temporary pacemaker backup should be prolonged up to day 6 after TASH in patients at increased risk (patients with retrograde atrioventricular block and at least 1 additional risk factor).

Purification of CK1 by affinity chromatography on immobilised axin.

Members of the Casein Kinase 1 (CK1) family are implicated in the regulation of a variety of physiological processes like development and circadian rhythm, as well as in diseases like cancer and Alzheimer's disease. From that perspective, CK1 family members are interesting targets for potential chemotherapy. We describe here a rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin. Axin is a scaffolding protein that interacts with a multitude of proteins, amongst them APC, GSK-3, beta-catenin, CK1alpha, delta, and epsilon, and PP2A. A GST-tagged axin peptide (residues 495-684) was produced in Escherichia coli and either immobilised on glutathione agarose beads or purified and immobilised on CNBr-activated sepharose 4B. These "GST-axin" matrices were found to selectively bind native CK1alpha and CK1epsilon from porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to efficiently purify large amounts of active native CK1 for screening purposes. This single step purification method also provides a convenient tool to follow the status of the axin-binding CK1 isoforms alpha, delta, and epsilon (protein levels, composition of isoforms, kinase activity) under different physiological settings.

Improved tumor control through circadian clock induction by Seliciclib, a cyclin-dependent kinase inhibitor.

The circadian timing system and the cell division cycle are frequently deregulated in cancer. The therapeutic relevance of the reciprocal interactions between both biological rhythms was investigated using Seliciclib, a cyclin-dependent kinase (CDK) inhibitor (CDKI). Mice bearing Glasgow osteosarcoma received Seliciclib (300 mg/kg/d orally) or vehicle for 5 days at Zeitgeber time (ZT) 3, 11, or 19. On day 6, tumor mRNA 24-hour expression patterns were determined for clock genes (Per2, Rev-erbalpha, and Bmal1) and clock-controlled cell cycle genes (c-Myc, Wee1, cyclin B1, and CDK1) with quantitative reverse transcription-PCR. Affinity chromatography on immobilized Seliciclib identified CDK1/CDK2 and extracellular signal-regulated kinase (ERK) 1/ERK2, CDK7/CDK9, and casein kinase CK1epsilon as Seliciclib targets, which respectively regulate cell cycle, transcription, and circadian clock in Glasgow osteosarcoma. Seliciclib reduced tumor growth by 55% following dosing at ZT3 or ZT11 and by 35% at ZT19 compared with controls (P < 0.001). Tolerability was also best at ZT3. Mean transcriptional activity of Rev-erbalpha, Per2, and Bmal1 was arrhythmic in the tumors of untreated mice. Seliciclib induced rhythmic clock gene expression patterns with physiologic phase relations only after ZT3 dosing. c-Myc and Wee1 mRNAs displayed synchronous circadian rhythms in the tumors of control mice receiving vehicle only but not in those of mice given the drug. Seliciclib further enhanced Wee1 expression irrespective of dosing time, an effect that reinforced G(2)-M gating. Seliciclib also inhibited CK1epsilon, which determines circadian period length. The coordination of clock gene expression patterns in tumor cells was associated with best antitumor activity of Seliciclib. The circadian clock and its upstream regulators represent relevant targets for CDKIs.

Cell cycle control by oscillating regulatory proteins in Caulobacter crescentus.

Significant strides have been made in recent years towards understanding the molecular basis of cell cycle progression in the model bacterium Caulobacter crescentus. At the heart of cell cycle regulation is a multicomponent transcriptional feedback loop, governing the production of successive regulatory waves or pulses of at least three master regulatory proteins. These oscillating master regulators direct the execution of phase-specific events and, importantly, through intrinsic genetic switches not only determine the length of a given phase, but also provide the driving force that catapults the cell into the next stage of the cell cycle. The genetic switches act as fail safe mechanisms that prevent the cell cycle from relapsing and thus govern the ordered production and the periodicity of these regulatory waves. Here, we detail how the master regulators CtrA, GcrA and DnaA coordinate cell cycle progression and polar development in Caulobacter.

Roscovitine targets, protein kinases and pyridoxal kinase.

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.