Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage.

To explore the role of Dicer-dependent control mechanisms in B lymphocyte development, we ablated this enzyme in early B cell progenitors. This resulted in a developmental block at the pro- to pre-B cell transition. Gene-expression profiling revealed a miR-17 approximately 92 signature in the 3'UTRs of genes upregulated in Dicer-deficient pro-B cells; a top miR-17 approximately 92 target, the proapoptotic molecule Bim, was highly upregulated. Accordingly, B cell development could be partially rescued by ablation of Bim or transgenic expression of the prosurvival protein Bcl-2. This allowed us to assess the impact of Dicer deficiency on the V(D)J recombination program in developing B cells. We found intact Ig gene rearrangements in immunoglobulin heavy (IgH) and kappa chain loci, but increased sterile transcription and usage of D(H) elements of the DSP family in IgH, and increased N sequence addition in Igkappa due to deregulated transcription of the terminal deoxynucleotidyl transferase gene.

Chemical and morphological analysis of dentin irradiated by different high-power lasers: a systematic review.

PURPOSE: This systematic review provides an overview of the main chemical and morphological alterations generated on dentin by different high-power lasers' irradiation. METHODS: The review was registered in PROSPERO (CRD42023394164) and PRISMA guidelines were followed. The search strategy was conducted on MEDLINE (PubMed), Embase (Elsevier), and Web of Science (Clarivate) databases. The eligibility criteria were established according to the PICOS strategy, focusing on in vitro and ex vivo studies that assessed the chemical and morphological changes in dentin using five high-power lasers: Nd:YAG (1064 nm), Er:YAG (2940 nm), Er, Cr:YSGG (2780 nm), diode (980 nm), and CO2 (10,600 nm). Publication range was from 2010 to 2022. Data was summarized in tables and risk of bias was assessed by QUIN tool. RESULTS: The search resulted in 2255 matches and 57 studies composed the sample. The methods most used to assess the outcomes were scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and Raman. The studies presented "medium" and "low" risk of bias. The laser prevalently identified was the Er:YAG laser, associated with dentin ablation, absence of smear layer, and exposed tubules. The Nd:YAG laser generated vitreous surface and thermal damage, such as carbonization and cracks. The other lasers caused an irregular surface and no adverse thermal effects. Regarding the chemical structure, only the Er,Cr:YSGG laser caused collagen matrix reduction. The effects found were more intense with higher dosimetry. CONCLUSION: Evidence available indicates that the irradiation of dentin with high-power lasers are related to morphological outcomes favorable to adhesive restorative procedures, with minimal changes in collagen matrix and mineral content. However, those observations should be carried carefully by clinicians and more clinical trials regarding the association of high-power laser irradiation and restorative procedure longevity are needed.

Approaching the Traveling Tournament Problem with Randomized Beam Search.

The traveling tournament problem is a well-known sports league scheduling problem famous for its practical hardness. Given an even number of teams with symmetric distances between their venues, a double round-robin tournament has to be scheduled minimizing the total travel distances over all teams. We consider the most common constrained variant without repeaters and a streak limit of three, for which we study a beam search approach based on a state-space formulation guided by heuristics derived from different lower bound variants. We solve the arising capacitated vehicle routing subproblems either exactly for small- to medium-sized instances up to 18 teams or heuristically also for larger instances up to 24 teams. In a randomized variant of the search, we employ random team ordering and add small amounts of Gaussian noise to the nodes' guidance for diversification when multiple runs are performed. This allows for a simple yet effective parallelization of the beam search. A final comparison is done on the NL, CIRC, NFL, and GALAXY benchmark instances with 12 to 24 teams, for which we report a mean gap difference to the best known feasible solutions of 1.2% and five new best feasible solutions.

[New treatments for spinal muscular atrophy]. / Neue Therapien der spinalen Muskelatrophie.

5­q-associated spinal muscular atrophy (SMA) has so far been a causally untreatable disease, which leads to severe, progressive physical restrictions due to the loss of spinal motor neurons. However, the monogenetic cause of the relatively short coding "survival motor neuron" (SMN) 1 gene sequence and the presence of almost identical gene copies, the SMN2 genes, offer favorable conditions for the development of new therapeutic approaches. While previously only supportive and palliative therapies could be used, new disease-modifying drugs are now available for the first time. Nusinersen, an antisense oligonucleotide (ASO), is the first drug that has received approval in Germany to treat SMA. Further therapeutic approaches such as the so-called "small molecules" or the gene replacement therapy are currently still being tested in clinical studies or are already waiting for approval by the European Medicines Agency (EMA). In this article, the most important disease-modifying drugs of SMA, the associated studies and their challenges are presented.

Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution.

The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.

Percutaneous Stone Manipulation.

Percutaneous stone manipulation by direct ultrasound disintegration, extraction or chemolysis was done on 34 patients. A total of 15 patients presented with an operatively established nephrostomy, while percutaneous nephrostomy and subsequent dilation of the nephrostomy channel were done in 19. The rate of complete stone clearance was 19 of 20 stones after percutaneous nephrostomy and 8 of 16 stones in the group with an operatively established nephrostomy. The primary goal, to remove obstructing pelvic stones, was achieved in all cases. There were no untoward side effects, such as back pressure damage owing to flushing of the collecting system during ultrasound disintegration, or persistent infection. Complications in 3 patients were managed conservatively.

Efficacy and mechanism of action of volasertib, a potent and selective inhibitor of Polo-like kinases, in preclinical models of acute myeloid leukemia.

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.

Electroencephalographic evaluation of the effectiveness of blunt trauma to induce loss of consciousness for on-farm killing of chickens and turkeys.

Euthanasia of small numbers of birds in case of injury or other illness directly on the farm may be necessary for welfare reasons. This should be done without transportation of the moribund animals in order to minimize pain and distress. Blood loss has to be avoided to minimize the risk of contaminating the environment. Cervical dislocation in combination with a blunt trauma may be an appropriate way to achieve this aim but the bird's age and body weight may influence the practicability of this method in the field. In this study, we evaluated broilers, broiler breeders, and turkeys of different age groups and weights up to nearly 16 kg for the efficacy of blunt trauma to induce unconsciousness, allowing subsequent killing of the bird without pain. The effect of blunt trauma on the brain was determined by electroencephalography (EEG). Auditory evoked potentials (AEPs) were recorded for each animal. Convulsions or tonic seizures were observed in all investigated animals after blunt trauma, including strong wing movements, torticollis, and stretching of legs. The EEG results demonstrate that the blunt trauma induced by a single, sufficiently strong hit placed in the frontoparietal region of the head led to a reduction or loss of the AEP in all groups of birds. These results clearly indicate a loss of sensibility and induction of unconsciousness, which would allow painless killing of the birds immediately after the induction of the blunt trauma.

A novel Fc-engineered monoclonal antibody to CD37 with enhanced ADCC and high proapoptotic activity for treatment of B-cell malignancies.

The tetraspanin CD37 is widely expressed in B-cell malignancies and represents an attractive target for immunotherapy with mAbs. We have chimerized a high-affinity mouse Ab to CD37 and engineered the CH2 domain for improved binding to human Fcγ receptors. The resulting mAb 37.1 showed high intrinsic proapoptotic activity on malignant B cells accompanied by homotypic aggregation. Furthermore, the Ab-mediated high Ab-dependent cell-mediated cytotoxicity (ADCC) on lymphoma and primary CLL cells. mAb 37.1 strongly depleted normal B cells as well as spiked B-lymphoma cells in blood samples from healthy donors as well as malignant B cells in blood from CLL patients. In all assays, mAb 37.1 was superior to rituximab in terms of potency and maximal cell lysis. A single dose of mAb CD37.1 administered to human CD37-transgenic mice resulted in a reversible, dose-dependent reduction of peripheral B cells. In a Ramos mouse model of human B-cell lymphoma, administration of mAb 37.1 strongly suppressed tumor growth. Finally, a surrogate Fc-engineered Ab to macaque CD37, with in vitro proapoptotic and ADCC activities very similar to those of mAb 37.1, induced dose-dependent, reversible B-cell depletion in cynomolgus monkeys. In conclusion, the remarkable preclinical pharmacodynamic and antitumor effects of mAb 37.1 warrant clinical development for B-cell malignancies.

[Process-oriented cost calculation in interventional radiology. A case study]. / Prozessorientierte Kostenrechnung in der Interventionellen Radiologie. Eine Fallstudie.

PURPOSE: Currently used costing methods such as cost centre accounting do not sufficiently reflect the process-based resource utilization in medicine. The goal of this study was to establish a process-oriented cost assessment of percutaneous radiofrequency (RF) ablation of liver and lung metastases. MATERIAL AND METHODS: In each of 15 patients a detailed task analysis of the primary process of hepatic and pulmonary RF ablation was performed. Based on these data a dedicated cost calculation model was developed for each primary process. The costs of each process were computed and compared with the revenue for in-patients according to the German diagnosis-related groups (DRG) system 2010. RESULTS: The RF ablation of liver metastases in patients without relevant comorbidities and a low patient complexity level results in a loss of EUR 588.44, whereas the treatment of patients with a higher complexity level yields an acceptable profit. The treatment of pulmonary metastases is profitable even in cases of additional expenses due to complications. CONCLUSION: Process-oriented costing provides relevant information that is needed for understanding the economic impact of treatment decisions. It is well suited as a starting point for economically driven process optimization and reengineering. Under the terms of the German DRG 2010 system percutaneous RF ablation of lung metastases is economically reasonable, while RF ablation of liver metastases in cases of low patient complexity levels does not cover the costs.

Automatic, three-segment, MR-based attenuation correction for whole-body PET/MR data.

PURPOSE: The combination of positron emission tomography (PET) and magnetic resonance (MR) tomography in a single device is anticipated to be the next step following PET/CT for future molecular imaging application. Compared to CT, the main advantages of MR are versatile soft tissue contrast and its capability to acquire functional information without ionizing radiation. However, MR is not capable of measuring a physical quantity that would allow a direct derivation of the attenuation values for high-energy photons. METHODS: To overcome this problem, we propose a fully automated approach that uses a dedicated T1-weighted MR sequence in combination with a customized image processing technique to derive attenuation maps for whole-body PET. The algorithm automatically identifies the outer contour of the body and the lungs using region-growing techniques in combination with an intensity analysis for automatic threshold estimation. No user interaction is required to generate the attenuation map. RESULTS: The accuracy of the proposed MR-based attenuation correction (AC) approach was evaluated in a clinical study using whole-body PET/CT and MR images of the same patients (n = 15). The segmentation of the body and lung contour (L-R directions) was evaluated via a four-point scale in comparison to the original MR image (mean values >3.8). PET images were reconstructed using elastically registered MR-based and CT-based (segmented and non-segmented) attenuation maps. The MR-based AC showed similar behaviour as CT-based AC and similar accuracy as offered by segmented CT-based AC. Standardized uptake value (SUV) comparisons with reference to CT-based AC using predefined attenuation coefficients showed the largest difference for bone lesions (mean value ± standard variation of SUV(max): -3.0% ± 3.9% for MR; -6.5% ± 4.1% for segmented CT). A blind comparison of PET images corrected with segmented MR-based, CT-based and segmented CT-based AC afforded identical lesion detectability, but slight differences in image quality were found. CONCLUSION: Our MR-based attenuation correction method offers similar correction accuracy as offered by segmented CT. According to the specialists involved in the blind study, these differences do not affect the diagnostic value of the PET images.

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase.

Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rgammac(null) (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34(+) and CD45(+) cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.

Surface mu heavy chain signals down-regulation of the V(D)J-recombinase machinery in the absence of surrogate light chain components.

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo.

Fine-mapping of the cell-division cycle, notably the identification of mitotic kinase signaling pathways, provides novel opportunities for cancer-drug discovery. As a key regulator of multiple steps during mitotic progression across eukaryotic species, the serine/threonine-specific Polo-like kinase 1 (Plk1) is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types . Here, we report the discovery of a potent small-molecule inhibitor of mammalian Plk1, BI 2536, which inhibits Plk1 enzyme activity at low nanomolar concentrations. The compound potently causes a mitotic arrest and induces apoptosis in human cancer cell lines of diverse tissue origin and oncogenome signature. BI 2536 inhibits growth of human tumor xenografts in nude mice and induces regression of large tumors with well-tolerated intravenous dose regimens. In treated tumors, cells arrest in prometaphase, accumulate phosphohistone H3, and contain aberrant mitotic spindles. This mitotic arrest is followed by a surge in apoptosis, detectable by immunohistochemistry and noninvasive optical and magnetic resonance imaging. For addressing the therapeutic potential of Plk1 inhibition, BI 2536 has progressed into clinical studies in patients with locally advanced or metastatic cancers.

BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity.

PURPOSE: Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers. To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis. EXPERIMENTAL DESIGN: The first compound in this series, suitable for i.v. administration, has entered clinical development. To fully explore the potential of Polo-like kinase 1 inhibition in oncology, we have profiled additional compounds and now describe a novel clinical candidate. RESULTS: BI 6727 is a highly potent (enzyme IC(50) = 0.87 nmol/L, EC(50) = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high volume of distribution and a long terminal half-life in mice (V(ss) = 7.6 L/kg, t(1/2) = 46 h) and rats (V(ss) = 22 L/kg, t(1/2) = 54 h). Second, BI 6727 has physicochemical and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer. With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. CONCLUSION: These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clinical studies have been initiated.

Pixel switching of epitaxial Pd/YHx/CaF2 switchable mirrors

Exposure of rare-earth films to hydrogen can induce a metal-insulator transition, accompanied by pronounced optical changes. This 'switchable mirror' effect has received considerable attention from theoretical, experimental and technological points of view. Most systems use polycrystalline films, but the synthesis of yttrium-based epitaxial switchable mirrors has also been reported. The latter form an extended self-organized ridge network during initial hydrogen loading, which results in the creation of micrometre-sized triangular domains. Here we observe homogeneous and essentially independent optical switching of individual domains in epitaxial switchable mirrors during hydrogen absorption. The optical switching is accompanied by topographical changes as the domains sequentially expand and contract; the ridges block lateral hydrogen diffusion and serve as a microscopic lubricant for the domain oscillations. We observe the correlated changes in topology and optical properties using in situ atomic force and optical microscopy. Single-domain phase switching is not observed in polycrystalline films, which are optically homogeneous. The ability to generate a tunable, dense pattern of switchable pixels is of technological relevance for solid-state displays based on switchable mirrors.

Carotid plaque analysis: comparison of dual-source computed tomography (CT) findings and histopathological correlation.

PURPOSE: Plaque morphology is an important predictor of stroke risk and may also be a predictor of postoperative outcome after carotid endarterectomy (CEA). Thus, the purpose of our study was to evaluate the findings of preoperative dual-source computed tomography (DSCT) of carotid plaque morphology and correlate these findings with histopathological findings. MATERIAL AND METHODS: Thirty patients undergoing CEA due to neurological events and high-grade carotid artery stenosis were evaluated with DSCT for degree of stenosis following the North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria and for non-invasive plaque morphology prior to CEA. CT protocol was as follows (SOMATOM Definition, Siemens Medical Solutions, Forchheim, Germany): A dual-energy protocol was used with tube A (140 kV, 55 mA) and tube B (80 kV, 230 mA) with 2 x 64 x 0.6-mm collimation, pitch 0.65 and rotation time of 0.33 s. Histopathological work-up was performed on the surgically retrieved tissues. The findings from DSCT and histopathology were compared with respect to image quality and plaque composition (fatty plaque, mixed plaque and calcified plaque), were correlated with histological specimens and classified according to the American Heart Association (AHA) classification of atherosclerotic plaque. Pearson correlation and kappa statistics were performed. RESULTS: The image quality of DSCT was rated as 'excellent' in all the examinations. The mean degree of stenosis was quantified as 82%. The sensitivity of DSCT for the detection of calcification was 100% (standard deviation (SD) 0%, confidence interval (CI): 99-100). While the sensitivity for the detection of mixed plaques was 89% (SD 12%, CI: 79-98), it was 85% (SD 10%, CI: 76-92) for the detection of low-density fatty plaques. The mean degree of agreement was k=0.81. CONCLUSION: DSCT angiography of the carotid arteries is feasible and the evaluation of carotid plaque composition allows non-invasive assessment of different plaque components. This may have an impact on the non-invasive differentiation of vulnerable plaques.

CD4+ and CD8a+ PET imaging predicts response to novel PD-1 checkpoint inhibitor: studies of Sym021 in syngeneic mouse cancer models.

Predicting the outcome of immunotherapy is essential for efficient treatment. The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment. Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year. However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response. Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs). Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4+ and CD8a+ TILs in syngeneic mouse tumor models for preclinical studies. Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4+ and CD8a+ TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 (89Zr-DFO-CD4/89Zr-DFO-CD8a). Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021. Results: Syngeneic tumor models were characterized as "hot" or "cold" according to number of TILs determined by flow cytometry and IHC. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%. The optimal imaging time-point was 24 hours post-injection of ~1 MBq tracer with 30 µg non-labeled co-dose. Reduced tumor and spleen uptake of 89Zr-DFO-CD8a was observed in CD8a+ depleted mice and the uptake was comparable with that of isotype control (89Zr-DFO-IgG2b) confirming specificity. PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of 89Zr-DFO-CD4 and 89Zr-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively). The maximum 89Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a 89Zr-DFO-CD4 ratio >9 (p=0.0018). Conclusion: We developed 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4+ and CD8a+ status. These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor. We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.

Sym021, a promising anti-PD1 clinical candidate antibody derived from a new chicken antibody discovery platform.

Discovery of therapeutic antibodies is a field of intense development, where immunization of rodents remains a major source of antibody candidates. However, high orthologue protein sequence homology between human and rodent species disfavors generation of antibodies against functionally conserved binding epitopes. Chickens are phylogenetically distant from mammals. Since chickens generate antibodies from a restricted set of germline genes, the possibility of adapting the Symplex antibody discovery platform to chicken immunoglobulin genes and combining it with high-throughput humanization of antibody frameworks by "mass complementarity-determining region grafting" was explored. Hence, wild type chickens were immunized with an immune checkpoint inhibitor programmed cell death 1 (PD1) antigen, and a repertoire of 144 antibodies was generated. The PD1 antibody repertoire was successfully humanized, and we found that most humanized antibodies retained affinity largely similar to that of the parental chicken antibodies. The lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding, resulting in elevated T-cell cytokine production in vitro. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to the clinically approved PD1 antibodies pembrolizumab and nivolumab. Moreover, Sym021 bound human PD1 with a stronger affinity (30 pM) compared to nivolumab and pembrolizumab, while also cross-reacting with cynomolgus and mouse PD1. This enabled direct testing of Sym021 in the syngeneic mouse in vivo cancer models and evaluation of preclinical toxicology in cynomolgus monkeys. Preclinical in vivo evaluation in various murine and human tumor models demonstrated a pronounced anti-tumor effect of Sym021, supporting its current evaluation in a Phase 1 clinical trial. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CD, cluster of differentiation; CDC, complement-dependent cytotoxicity; CDR, complementarity determining region; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; FR, framework region; GM-CSF, granulocyte-macrophage colony-stimulating factor; HRP, horseradish peroxidase; IgG, immunoglobulin G; IL, interleukin; IFN, interferon; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; NK, natural killer; PBMC, peripheral blood mono-nuclear cell; PD1, programmed cell death 1; PDL1, programmed cell death ligand 1; RT-PCR, reverse transcription polymerase chain reaction; SEB, Staphylococcus Enterotoxin B; SPR, surface Plasmon Resonance; VL, variable part of light chain; VH, variable part of heavy chain.

Multipolar hepatic radiofrequency ablation using up to six applicators: preliminary results.

PURPOSE: To evaluate the clinical feasibility and safety of hepatic radiofrequency (RF) ablation using a multipolar RF system permitting the simultaneous use of up to six electrodes. MATERIALS AND METHODS: Ten patients (3 female, 7 male, mean age 61) suffering from 29 hepatic metastases (range: 1 - 5) of different tumors were treated with a modified multipolar RF system (CelonLab Power, Celon Medical Instruments, Teltow, Germany) operating four to six needle-shaped internally cooled RF applicators. The procedure duration, applied energy and generator output were recorded during the intervention. The treatment result and procedure-related complications were analyzed. The achieved coagulation volume was calculated on the basis of contrast-enhanced CT scans 24 hours after RF ablation. RESULTS: Complete tumor ablation was achieved in all cases as determined by the post-interventional lack of contrast enhancement in the target region using four applicators in five patients, five applicators in one patient and six applicators in four patients. A mean energy deposition of 353.9 +/- 176.2 kJ resulted in a mean coagulation volume of 115.9 +/- 79.5 cm (3). The mean procedure duration was 74.9 +/- 21.2 minutes. Four patients showed an intraabdominal hemorrhage which necessitated further interventional treatment (embolization; percutaneous histoacryl injection) in two patients. CONCLUSION: Multipolar RF ablation of hepatic metastasis with up to six applicators was clinically feasible. In our patient population it was associated with an increased risk of intraabdominal bleeding probably due to the multiple punctures associated with the use of multiple applicators.