Evaluation of the anti-inflammatory activity of Acacia polyphylla and identification of a new apigenin-3-C- glycosylated type flavonoid.

Owing to the potentially harmful adverse effects of current anti-inflammatory drugs, there is a need to identify new alternative substances. Thus, this study aimed to perform a phytochemical analysis of A. polyphylla to identify compounds responsible for its anti-inflammatory activity. Several fractions of the A. polyphylla extract were obtained and evaluated in an ex vivo anti-inflammatory assay using fresh human blood. Among the evaluated fractions, the BH fraction displayed the highest percentage of PGE2 inhibition (74.8%) compared to the reference drugs dexamethasone and indomethacin, demonstrating its excellent potential for anti-inflammatory activity. Astragalin (P1), a known 3-O-glucoside of kaempferol, was isolated from the A. polyphylla extract for the first time. In addition, a new compound (P2) was isolated and identified as the apigenin-3-C-glycosylated flavonoid. Astragalin showed moderate PGE2 activity (48.3%), whereas P2 was not anti-inflammatory. This study contributes to the phytochemical studies of A. polyphylla and confirms its anti-inflammatory potential.

Vasodilation promoted by (E,E)-farnesol involving ion channels in human umbilical arteries.

Background: (E,E)-farnesol is a sesquiterpene alcohol derived from plants and animals that exhibits pharmacological properties in the cardiovascular system. However, its effects on human umbilical vessels remain unknown. Purpose: Thus, this study aims to characterize the vasodilatory effect of (E,E)-farnesol in human umbilical arteries (HUA). Study design: The tissue is obtained from pregnant women over 18 years of age, normotensive, and without prepartum complications. After collected, the tissue was segmented and dissected to remove Wharton's jelly and obtain the umbilical arteries segments. Methods: HUA segments were isolated and sectioned into rings that were subjected to isometric tension recordings in an organ bath. Results: (E,E)-farnesol (1 µmol/L to 1 mmol/L) promoted vasodilatory effect in HUA preparations, affecting basal tone, and inhibiting the electromechanical coupling induced by KCl 60 mmol/L with greater potency (EC50 225.3 µmol/L) than the pharmacomechanical coupling induced by 5-HT 10 µmol/L (EC50 363.5 µmol/L). In the absence of extracellular calcium, pharmacomechanical coupling was also abolished, and contractions induced by CaCl2 or BaCl2 were attenuated by (E,E)-farnesol indicating a possible direct inhibition of L-type VOCC as a mechanism of the vasodilatory effect. The vasodilator efficacy of (E,E)-farnesol on reduction of vasocontraction induced by the presence of tetraethylammonium (1 or 10 mmol/L), 4-aminopyridine (1 mmol/L) and glibenclamide (10 µmol/L) suggesting a possible influence of different potassium channels (BKCa, KV and KATP). Conclusion: These results suggest that (E,E)-farnesol may be a promising pharmacological candidate for obstetric hypertensive disorders.

Temozolomide and Capecitabine Treatment for an Aggressive Somatotroph Pituitary Tumor: A Case Report and Literature Review.

Aggressive somatotroph pituitary tumor that causes acromegaly is extremely rare and resists conventional treatments such as multiple surgeries, radiotherapies, and various types of somatostatin analogs. Here, we propose a novel treatment option for these rare cases by discussing our case and reviewing the literature. We experienced an aggressive somatotroph tumor in a 52-year-old woman with acromegaly. Not only could a complete remission of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) not be obtained, but the tumor continued to grow and eventually recurred around the brainstem despite multidisciplinary treatments. We employed immunohistochemistry and a three-dimensional (3D) spheroid ex vivo assay to determine the best treatment option for this case. Although histology showed strong O 6-methylguanine DNA methyltransferase expression and high Ki-67 labeling index (22%), temozolomide (TMZ) combined with capecitabine (CAPTEM) treatment was performed based on the results of the patient-derived 3D spheroid ex vivo assay, which predicted more effective treatment with CAPTEM than with TMZ alone. Consequently, GH and IGF-1 levels were restored to normal range with remarkable tumor shrinkage after CAPTEM treatment. To the best of our knowledge, there have been even very few reports describing successful treatment for such aggressive and refractory somatotroph tumors and this is the first report showing the effectiveness of CAPTEM on refractory somatotroph tumor both ex vivo and in vivo.

A high-throughput assay for screening the abilities of per- and polyfluoroalkyl substances in inducing plasma kallikrein-like activity.

The plasma consists of multiple functional serine zymogens, such as plasma kallikrein-kinin system (KKS), which are vulnerable to exogenous chemical exposure, and may closely relate to the deleterious effects. Testing whether the anthropogenic chemicals could increase the kallikrein-like activity in plasma or not would be of great help to understand their potentials in triggering the cascade activation of the plasma zymogens and explain the corresponding hematotoxicity. In this study, a novel high-throughput ex vivo assay was established to screen the abilities of emerging chemicals like per- and polyfluoroalkyl substances (PFASs) in inducing kallikrein-like activities on basis of using rat plasma as the protease zymogen source. Upon the optimization of the conditions in the test system, the assay gave sensitive fluorescent response to the stimulation of the positive control, dextran sulfate, and the dose-response showed a typical S-shaped curve with EC50 of 0.24 mg/L. The intra-plate and inter-plate relative standard deviations (RSDs) were less than 10% in the quantitative range of dextran sulfate, indicating a good reliability and repeatability of this newly-established assay. Using this method, several alternatives or congeners of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), including 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFESA), Ag-PFOA, K-PFOA, Na-PFOA and ammonium pentadecafluorooctanoate (APFO), were further screened, and their capabilities in inducing kallikrein-like activities were identified. The ex vivo assay newly-developed in the present study would be promising in high-throughput screening of the hematological effects of emerging chemicals of concern.

Biomarkers of geno- and cytotoxicity in the native broad-snouted caiman (Caiman latirostris): Chromosomal aberrations and mitotic index.

We evaluated the sensitivity of the chromosomal aberration (CA) and mitotic index (MI) assays on peripheral blood lymphocytes (PBLs) of Caiman latirostris, following ex vivo exposure to the alkylating agent, MMS. Two concentrations of MMS were tested in cultured peripheral blood. Relative to controls, MMS exposure reduced the number of metaphases observed, but both the numbers of cells with MN and the percentages of aberrant metaphases increased. The types of CA identified were chromosome and chromatid breaks, chromosomal rearrangements, monosomies, and nullisomies, with significantly higher values in the MMS-exposed groups. The incorporation of the MI and CA tests in C. latirostris can provide information on damage caused by xenobiotic exposures.

Construction of in vitro patient-derived tumor models to evaluate anticancer agents and cancer immunotherapy.

An in vitro assay system using patient-derived tumor models represents a promising preclinical cancer model that replicates the disease better than traditional cell culture models. Patient-derived tumor organoid (PDO) and patient-derived tumor xenograft (PDX) models have been previously established from different types of human tumors to recapitulate accurately and efficiently their tissue architecture and function. However, these models have low throughput and are challenging to construct. Thus, the present study aimed to establish a simple in vitro high-throughput assay system using PDO and PDX models. Furthermore, the current study aimed to evaluate different classes of anticancer drugs, including chemotherapeutic, molecular targeted and antibody drugs, using PDO and PDX models. First, an in vitro high-throughput assay system was constructed using PDO and PDX established from solid and hematopoietic tumors cultured in 384-well plates to evaluate anticancer agents. In addition, an in vitro evaluation system of the immune response was developed using PDO and PDX. Novel cancer immunotherapeutic agents with marked efficacy have been used against various types of tumor. Thus, there is an urgent need for in vitro functional potency assays that can simulate the complex interaction of immune cells with tumor cells and can rapidly test the efficacy of different immunotherapies or antibody drugs. An evaluation system for the antibody-dependent cellular cytotoxic activity of anti-epidermal growth factor receptor antibody and the cytotoxic activity of activated lymphocytes, such as cytotoxic T lymphocytes and natural killer cells, was constructed. Moreover, immune response assay systems with bispecific T-cell engagers were developed using effector cells. The present results demonstrated that in vitro assay systems using PDO and PDX may be suitable for evaluating anticancer agents and immunotherapy potency with high reproducibility and simplicity.

Mitigation of silver nanoparticle toxicity by humic acids in gills of Piaractus mesopotamicus fish.

Silver nanoparticles (AgNPs) are one of the most produced nanoproducts due to their unique biocide properties. The natural organic matter has an important impact on nanoparticle's dispersion as it may alter their fate and transport, as well as their bioavailability and toxicity. Therefore, this study aimed to evaluate the mitigatory effect of humic acids (HAs) on AgNP toxicity. For this purpose, we carried out an ex vivo exposure of gill of Piaractus mesopotamicus fish to 100 µg L-1 of AgNPs or AgNO3, alone and in combination with 10 mg L-1 of HAs. In parallel, a complete AgNP characterization in the media, including the presence of HAs, was provided, and the Ag+ release was measured. We analyzed Ag bioaccumulation, antioxidant enzymes activities, lipid peroxidation, antioxidant capacity against peroxyl radicals, and reduced glutathione levels in fish tissue. Our results indicated the Ag+ release from AgNPs decreased 28% when the HAs were present in the media. The Ag accumulation in gill tissue exposed to AgNPs alone was higher than the AgNO3 exposure, and sixfold higher than the treatment with the HA addition. Moreover, after both Ag forms, the catalase enzyme augmented its activity. However, those responses were mitigated when the HAs were present in the media. Then, our results suggested the mitigation by HAs under the exposure to both Ag forms, providing valuable information about the fate and behavior of this emergent pollutant.

Molecular Studies and an ex vivo Complement Assay on Endothelium Highlight the Genetic Complexity of Atypical Hemolytic Uremic Syndrome: The Case of a Pedigree With a Null CD46 Variant.

Atypical hemolytic uremic syndrome (aHUS) is an ultra-rare disease characterized by microangiopathic hemolysis, thrombocytopenia, and renal impairment and is associated with dysregulation of the alternative complement pathway on the microvascular endothelium. Outcomes have improved greatly with pharmacologic complement C5 blockade. Abnormalities in complement genes (CFH, CD46, CFI, CFB, C3, and THBD), CFH-CFHR genomic rearrangements, and anti-FH antibodies have been reported in 40-60% of cases. The penetrance of aHUS is incomplete in carriers of complement gene abnormalities; and multiple hits, including the CFH-H3 and CD46 GGAAC risk haplotypes and the CFHR1 * B risk allele, as well as environmental factors, contribute to disease development. Here, we investigated the determinants of penetrance of aHUS associated with CD46 genetic abnormalities. We studied 485 aHUS patients and found CD46 rare variants (RVs) in about 10%. The c.286+2T>G RV was the most prevalent (13/485) and was associated with <30% penetrance. We conducted an in-depth study of a large pedigree including a proband who is heterozygous for the c.286+2T>G RV who experienced a severe form of aHUS and developed end-stage renal failure. The father and paternal uncle with the same variant in homozygosity and six heterozygous relatives are unaffected. Flow cytometry analysis showed about 50% reduction of CD46 expression on blood mononuclear cells from the heterozygous proband and over 90% reduction in cells from the proband's unaffected homozygous father and aunt. Further genetic studies did not reveal RVs in known aHUS-associated genes or common genetic modifiers that segregated with the disease. Importantly, a specific ex vivo test showed excessive complement deposition on endothelial cells exposed to sera from the proband, and also from his mother and maternal uncle, who do not carry the c.286+2T>G RV, indicating that they share a circulating defect that results in complement dysregulation on the endothelium. These results highlight the complexity of the genetics of aHUS and indicate that CD46 deficiency may not be enough to induce aHUS. We hypothesize that the proband inherited from his mother a genetic abnormality in a complement circulating factor that has not been identified yet, which synergized with the CD46 RV in predisposing him to the aHUS phenotype.

Organotypic Culture Assay for Neuromuscular Synaptic Degeneration and Function.

We describe here an organotypic culture system we have used to investigate mechanisms that maintain structure and function of axon terminals at the neuromuscular junction (NMJ). We developed this by taking advantage of the slow Wallerian degeneration phenotype in mutant Wlds mice, using these to compare preservation of NMJs with degeneration in nerve-muscle preparations from wild-type mice. We take hind limb tibial nerve/flexor digitorum brevis and lumbrical muscles and incubate them in mammalian physiological saline at 32 °C for 24-48 h. Integrity of NMJs can then be compared using a combination of electrophysiological and morphological techniques. We illustrate our method with data showing synaptic preservation ex vivo in nerve-muscle explants from Sarm-1 null-mutant mice. The ex vivo assays of NMJ integrity we describe here may therefore be useful for detailed investigation of synaptic maintenance and degeneration.

Antioxidant Properties of Sourdoughs Made with Whole Grain Flours of Hull-Less Barley or Conventional and Pigmented Wheat and by Selected Lactobacilli Strains.

The use of sourdough fermentation and whole grain flours in baked goods manufacturing are known to enhance their functional and nutritional features. In this context, it is necessary to select the most suitable lactic acid bacteria strains and flour combination to achieve this goal. A characterization of 70 lactobacilli strains based on pro-technological and nutritional properties was carried out. The screening allowed the selection of 10 strains that were used to ferment sourdoughs made with two varieties of common wheat, the conventional red-grained cv Aubusson, a blue-grained variety rich in anthocyanins cv Skorpion, and a hull-less barley variety, cv Rondo. From each fermented sourdough, a water soluble extract was obtained and evaluated for its antioxidant activity performed on cultured cells (RAW 264.7 murine macrophage) by assaying Reactive Oxygen Species (ROS) content. Sourdoughs made with pigmented wheat and barley, had an antioxidant activity greater than that recovered in those made with conventional wheat flour, in spite they have been inoculated with the same LAB strains. Results highlighted the interdependence between flour and the inoculated lactic acid bacteria that has to be taken into account for the development of healthy breads exploiting high functional value cereals through biotechnological processes.

A functional ex vivo assay to detect PARP1-EJ repair and radiosensitization by PARP-inhibitor in prostate cancer.

Here, we present a functional assay to detect the repair switch to the alternative PARP1-dependent end joining (PARP1-EJ) pathway and the associated susceptibility to PARPi-mediated radiosensitization in freshly collected tumor samples from prostate cancer (PCa) patients, thereby facilitating the selection of patients who should benefit from combined PARPi plus radiotherapy (RT) treatment. Our optimized ex-vivo approach sustains tumor slices for up to 15 days under culture conditions that maintain proliferation and oxygenation rates, as measured by EdU incorporation and pimonidazole staining, respectively. We present a robust system to analyze DSB repair using, for the first time in an ex vivo tumor slice setting, two DSB-markers simultaneously i.e. γH2AX and 53BP1. A computer-based processing method (i) controls variations in DNA content and slicing on the number of repair foci and (ii) measures the PARPi-mediated enhancement ratio on DSB foci numbers to ensure inter-patient-comparability. We validated this approach using a PC3 xenograft model with its previously described repair switch to PARP1-EJ. More importantly, we show that approximately 30% of the analyzed tumor tissue samples collected from PCa patients display a switch to PARP1-EJ, as indicated by the enhanced number of residual γH2AX/53BP1 foci exclusively after PARPi+RT. Furthermore, normal prostatic tissues show no repair switch to PARP1-EJ, indicating that this repair switch and its associated radiosensitizing effect is tumor-specific. Collectively, we present here a predictive assay for the switch to PARP1-EJ that enables individualization of anti-cancer treatment using a combination of RT and radiosensitizing anticancer agents such as PARPi in PCa.

A precision medicine test predicts clinical response after idarubicin and cytarabine induction therapy in AML patients.

Complete remission (CR) after induction therapy is the first treatment goal in acute myeloid leukemia (AML) patients and has prognostic impact. Our purpose is to determine the correlation between the observed CR/CRi rate after idarubicin (IDA) and cytarabine (CYT) 3 + 7 induction and the leukemic chemosensitivity measured by an ex vivo test of drug activity. Bone marrow samples from adult patients with newly diagnosed AML were included in this study. Whole bone marrow samples were incubated for 48 h in well plates containing IDA, CYT, or their combination. Pharmacological response parameters were estimated using population pharmacodynamic models. Patients attaining a CR/CRi with up to two induction cycles of 3 + 7 were classified as responders and the remaining as resistant. A total of 123 patients fulfilled the inclusion criteria and were evaluable for correlation analyses. The strongest clinical predictors were the area under the curve of the concentration response curves of CYT and IDA. The overall accuracy achieved using MaxSpSe criteria to define positivity was 81%, predicting better responder (93%) than non-responder patients (60%). The ex vivo test provides better yet similar information than cytogenetics, but can be provided before treatment representing a valuable in-time addition. After validation in an external cohort, this novel ex vivo test could be useful to select AML patients for 3 + 7 regimen vs. alternative schedules.

Lanosterol and 25-hydroxycholesterol dissociate crystallin aggregates isolated from cataractous human lens via different mechanisms.

Cataract, a crystallin aggregation disease, is the leading cause of human blindness worldwide. Surgery is the only established treatment of cataracts and no anti-cataract drugs are available thus far. Recently lanosterol and 25-hydroxycholesterol have been reported to redissolve crystallin aggregates and partially restore lens transparency in animals. However, the efficacies of these two compounds have not been quantitatively studied ex vivo using patient tissues. In this research, we developed a quantitative assay applicable to efficacy validations and mechanistic studies by a protocol to isolate protein aggregates from the surgically removed cataractous human lens. Our results showed that both compounds were effective for human cataractous samples with EC50 values at ten micromolar level. The efficacies of both compounds strongly depended on cataract severity. Lanosterol and 25-hydroxycholesterol were two mechanistically different lead compounds of anti-cataract drug design.

Measuring ex vivo drug susceptibility in Plasmodium vivax isolates from Cambodia.

BACKGROUND: While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013-2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. RESULTS: Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013-4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. CONCLUSION: The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method.

Molecularly targeted drug combinations demonstrate selective effectiveness for myeloid- and lymphoid-derived hematologic malignancies.

Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.

Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis.

Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.

YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network.

Specific overexpression in cancer cells and evidence of oncogenic functions make Survivin an attractive target in cancer therapy. The small molecule compound YM155 has been described as the first "Survivin suppressant" but molecular mechanisms involved in its biological activity and its clinical potential remain obscure. We herein show that YM155 exerts single agent toxicity on primary breast cancer cells grown in an ex vivo assay preserving tumor microenvironment. In vitro assays indicate that YM155 more efficiently triggers cell death in breast cancer cells (including these with stem-cell like properties) than in non tumorigenic mammary cells. YM155-induced cell death is critically dependent on autophagy and NF-kB but independent of p53 and it coïncides with DNA damage and a DNA damage response in p53-proficient cells. Our results point out a crosstalk between NF-kB and autophagy controlling YM155-induced death in breast cancer cells and argue for the potential use of YM155 as a genotoxic agent in breast cancer therapy.

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library.

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.