Exploring the Potentiality of a Plant Platform for Monoclonal Antibody Production in Veterinary Medicine.

Canine atopic dermatitis (CAD) is an allergic, inflammatory, and pruritic skin disease associated with the production of IgE antibodies against environmental allergens and mainly house dust mite allergens. This complex dermatological pathology involves Interleukin 31 (IL-31) as a central itch mediator. One of the most effective CAD treatments is a caninized monoclonal antibody (mAb) called Lokivetmab. It is produced in CHO cells and targets specifically canine IL-31 (cIL-31) and blocks its cellular messaging. This treatment has undoubtedly contributed to a breakthrough in dermatitis-related pruritus. However, its production in mammalian cells requires time-consuming procedures, high production costs, and investment. Plants are considered an emerging protein production platform for recombinant biopharmaceuticals due to their cost-effectiveness and rapidity for production. Here, we use transient expression in Nicotiana benthamiana plants to produce recombinant canine Interleukin 31 (cIL-31) and an anti-IL-31 monoclonal antibody (M1). First, we describe the production and characterization of M1 and then its activity on an IL-31-induced pruritic model in dogs compared to its commercial homolog. Dogs treated with the plant-made M1 mAb have shown similar improvements to Lokivetmab-treated ones after different challenges using canine IL-31. Furthermore, M1 injections were not associated with any side effects. These results demonstrate the safety and efficacy of this plant-made Lokivetmab biosimilar to control dogs' pruritus in a well-established model. Finally, this study shows that the plant-production platform can be utilized to produce rapidly functional mAbs and bring hope to the immunotherapy field of veterinary medicine.

A fast and easy one-step purification strategy for plant-made antibodies using Protein A magnetic beads.

A major difficulty to reach commercial- scale production for plant-made antibodies is the complexity and cost of their purification from plant extracts. Here, using Protein A magnetic beads, two monoclonal antibodies are purified in a one-step procedure directly from non-clarified crude plant extracts. This technique provides significant savings in terms of resources, operation time, and equipment.

Rapid Conversion of Amyloid-Beta 1-40 Oligomers to Mature Fibrils through a Self-Catalytic Bimolecular Process.

The formation of fibrillar aggregates of the amyloid beta peptide (Aß) in the brain is one of the hallmarks of Alzheimer's disease (AD). A clear understanding of the different aggregation steps leading to fibrils formation is a keystone in therapeutics discovery. In a recent study, we showed that Aß40 and Aß42 form dynamic micellar aggregates above certain critical concentrations, which mediate a fast formation of more stable oligomers, which in the case of Aß40 are able to evolve towards amyloid fibrils. Here, using different biophysical techniques we investigated the role of different fractions of the Aß aggregation mixture in the nucleation and fibrillation steps. We show that both processes occur through bimolecular interplay between low molecular weight species (monomer and/or dimer) and larger oligomers. Moreover, we report here a novel self-catalytic mechanism of fibrillation of Aß40, in which early oligomers generate and deliver low molecular weight amyloid nuclei, which then catalyze the rapid conversion of the oligomers to mature amyloid fibrils. This fibrillation catalytic activity is not present in freshly disaggregated low-molecular weight Aß40 and is, therefore, a property acquired during the aggregation process. In contrast to Aß40, we did not observe the same self-catalytic fibrillation in Aß42 spheroidal oligomers, which could neither be induced to fibrillate by the Aß40 nuclei. Our results reveal clearly that amyloid fibrillation is a multi-component process, in which dynamic collisions between different interacting species favor the kinetics of amyloid nucleation and growth.

[Amoxicillin induced crystal nephropathy : Monitoring of residual plasma levels]. / Cristalluries sous amoxicilline : intérêt du monitoring des concentrations plasmatiques résiduelles.

Since 2010, a lot of cases of amoxicillin induced crystal nephropathy have been reported to the French pharmacovigilance centers partly due to the high doses recommended by infectious disease guidelines. Typical clinical presentation and exclusion of others toxics or immuno-allergic causes are mandatory to assess the diagnostic. Amoxicillin crystals are rarely found or searched and renal biopsy is not frequently performed due to technical reasons and prompt renal recovery after antibiotics withdrawal. Monitoring of residual plasma concentration is rarely used in clinical practice for diagnostic or prognostic interest. We present 9 consecutive cases of acute kidney injury suspected to be due to amoxicillin crystals with residuals plasma levels to disclose a predictive threshold of tubulopathy. All patients had a high residual rate at diagnosis but we cannot find a threshold that would allow to adapt the antibiotic dose, enhance hydratation and alkalinizide urine to increase the medication solubility and limit renal toxicity.

Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors.

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins.

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.

Combining Structural Aggregation Propensity and Stability Predictions To Redesign Protein Solubility.

The aggregation propensity of each particular protein seems to be shaped by evolution according to its natural abundance in the cell. The production and downstream processing of recombinant polypeptides implies attaining concentrations that are orders of magnitude above their natural levels, often resulting in their aggregation; a phenomenon that precludes the marketing of many globular proteins for biomedical or biotechnological applications. Therefore, there is a huge interest in methods aimed to increase the proteins solubility above their natural limits. Here, we demonstrate that an updated version of our AGGRESCAN 3D structural aggregation predictor, that now takes into account protein stability, allows for designing mutations at specific positions in the structure that improve the solubility of proteins without compromising their conformation. Using this approach, we have designed a highly soluble variant of the green fluorescent protein and a human single-domain VH antibody displaying significantly reduced aggregation propensity. Overall, our data indicate that the solubility of unrelated proteins can be easily tuned by in silico-designed nondestabilizing amino acid changes at their surfaces.

Dynamic micellar oligomers of amyloid beta peptides play a crucial role in their aggregation mechanisms.

A deep understanding of the early molecular mechanism of amyloid beta peptides (Aß) is crucial to develop therapeutic and preventive approaches for Alzheimer's disease (AD). Using a variety of biophysical techniques, we have found that micelle-like dynamic oligomers are rapidly formed by Aß40 and Aß42 above specific critical concentrations. Analysis of the initial aggregation rates at 37 °C measured by thioflavin T and Bis-ANS fluorescence using a mass-action micellization model revealed a concentration-dependent switch in the nucleation mechanism. Bimolecular nucleation appears to occur at low peptide concentration while above the critical micellar concentration, the nucleation takes place more efficiently in the micelles. Upon incubation, these micelles mediate a rapid formation of larger, more stable oligomers enriched in beta-sheet structure. These oligomers formed from Aß40, enriched in amyloid nuclei, acquire a higher capacity to fibrillate than their micellar precursors. Aß42 can also form similar oligomers but they have lower beta-sheet structure content and lower capacity to fibrillate. On the other hand, a considerable fraction of the Aß42 peptide forms morphologically distinct oligomers that are unable to fibrillate and show significant effect on SH-SY5Y cell viability. Overall, our results highlight the importance of micellar structures as mediators of amyloid nucleation and contribute to the understanding of the differences between the aggregation pathways of Aß40 and Aß42.

A mechanism for cancer-associated inactivation of NQO1 due to P187S and its reactivation by the consensus mutation H80R.

The cancer-associated P187S polymorphism in the NAD(P)H:quinone oxidoreductase 1 (NQO1) abolishes enzyme activity by strongly reducing FAD binding affinity. A single mammalian consensus mutation (H80R) protects P187S from inactivation. To provide mechanistic insight into these effects, we report here a detailed structural and thermodynamic characterization of FAD binding to these NQO1 variants. Our results show that H80R causes a population shift in the conformational ensemble of apo-P187S, remodelling the structure and dynamics of the FAD-binding site and reducing the energetic penalization arising from the equilibrium between apo- and holo-states. Our analyses illustrate how single amino acid changes can profoundly affect structural and mechanistic features of protein functional traits, with implications for our understanding of protein evolution and human disease.

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development.

UNLABELLED: The remarkable metal resistance of many microorganisms is related to the presence of multiple metal resistance operons. Pseudomonas putida KT2440 can be considered a model for these microorganisms since its arsenic resistance is due to the action of proteins encoded by the two paralogous arsenic resistance operons ARS1 and ARS2. Both operons contain the genes encoding the transcriptional regulators ArsR1 and ArsR2 that control operon expression. We show here that purified ArsR1 and ArsR2 bind the trivalent salt of arsenic (arsenite) with similar affinities (~30 µM), whereas no binding is observed for the pentavalent salt (arsenate). Furthermore, trivalent salts of bismuth and antimony showed binding to both paralogues. The positions of cysteines, found to bind arsenic in other homologues, indicate that ArsR1 and ArsR2 employ different modes of arsenite recognition. Both paralogues are dimeric and possess significant thermal stability. Both proteins were used to construct whole-cell, lacZ-based biosensors. Whereas responses to bismuth were negligible, significant responses were observed for arsenite, arsenate, and antimony. Biosensors based on the P. putida arsB1 arsB2 arsenic efflux pump double mutant were significantly more sensitive than biosensors based on the wild-type strain. This sensitivity enhancement by pump mutation may be a convenient strategy for the construction of other biosensors. A frequent limitation found for other arsenic biosensors was their elevated background signal and interference by inorganic phosphate. The constructed biosensors show no interference by inorganic phosphate, are characterized by a very low background signal, and were found to be suitable to analyze environmental samples. IMPORTANCE: Arsenic is at the top of the priority list of hazardous compounds issued by the U.S. Agency for Toxic Substances and Disease. The reason for the stunning arsenic resistance of many microorganisms is the existence of paralogous arsenic resistance operons. Pseudomonas putida KT2440 is a model organism for such bacteria, and their duplicated ars operons and in particular their ArsR transcription regulators have been studied in depth by in vivo approaches. Here we present an analysis of both purified ArsR paralogues by different biophysical techniques, and data obtained provide valuable insight into their structure and function. Particularly insightful was the comparison of ArsR effector profiles determined by in vitro and in vivo experimentation. We also report the use of both paralogues to construct robust and highly sensitive arsenic biosensors. Our finding that the deletion of both arsenic efflux pumps significantly increases biosensor sensitivity is of general relevance in the biosensor field.

High specificity in CheR methyltransferase function: CheR2 of Pseudomonas putida is essential for chemotaxis, whereas CheR1 is involved in biofilm formation.

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (KD of 0.14-2.2 µM) than the substrate S-adenosylmethionine (KD of 22-43 µM), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.

Characterization of oligomers of heterogeneous size as precursors of amyloid fibril nucleation of an SH3 domain: an experimental kinetics study.

Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.

Evaluation of a new disinfection method for ultrasound probes used for regional anesthesia: ultraviolet C light.

OBJECTIVES: Ultrasound-guided regional anesthesia is commonly used for block placement. At present, the risk of cross contamination from probes is not well documented. To avoid transmission of infectious agents, several methods have been used for probe disinfection and protection. The aim of this study was to evaluate the antibacterial efficacy of a new high-level disinfection method based on ultraviolet C (UV-C) light under routine conditions after block placement with an unprotected probe. METHODS: The study was after approval by the local Ethics Committee. In the first part of the study, 15 ultrasound probes were exposed to a large inoculum of 3 bacteria. Ultraviolet C disinfection consisted of cleaning the probe with dry and disinfectant-impregnated paper followed by a 90-second UV-C disinfection cycle in a decontamination chamber. A protocol was established to retrieve the probe with sterile gloves after opening the door of the chamber. In the second part, 50 blocks were placed with ultrasound-guided regional anesthesia. The skin was first prepared with an antiseptic solution, and sterile gel was applied; no covers were used to protect the probes. The blocks were then disinfected with UV-C light. Bacteriologic samples were collected before and after the UV-C method and inoculated on chocolate agar plates. RESULTS: During the first part of the study, all probes were infected after inoculation (>150 colony-forming units) but were considered sterile (<10 colony-forming units) after disinfection. During the second part of the study, all probes were considered sterile before and after disinfection. CONCLUSIONS: Ultraviolet C disinfection seems relevant for ultrasound-guided regional anesthesia just before block placement. It offers simple, fast, and effective high-level disinfection. Moreover, this method should obviate the use of sterile probe covers, which can improve echogenicity.

Comfort of the patient during axillary blocks placement: a randomized comparison of the neurostimulation and the ultrasound guidance techniques.

BACKGROUND: Axillary brachial plexus block under neurostimulation is commonly used for upper limb surgery, but it is sometimes recognized as an uncomfortable technique, with most patients identifying electrical stimulation as an unpleasant moment. Ultrasound-guided regional anaesthesia, which becomes an increasingly popular technique, does not require electrical stimulation and then should theoretically improve axillary block placement comfort. The aim of this study was to compare the comfort of the patients during axillary block placement with neurostimulation and ultrasound guidance using either the out-of-plane or the in-plane approach. METHODS: Consecutive patients were prospectively enrolled in three equal groups: neurostimulation, ultrasound out-of-plane and ultrasound in-plane approaches. A score was used to measure the comfort of the patients during axillary blocks placement. This score included three criteria: maximum pain intensity perceived during block placement measured using a visual analogue scale (0, no pain and 100, maximal or worse imaginable pain), the number of unpleasant events declared by the patients and the satisfaction of the patient (unsatisfied, acceptable, satisfied, very satisfied). The comfort score was calculated as the sum of each criterion, which was attributed a value of 0 or 1: visual analogue scale (30/100, 0), number of unpleasant events (0, 1; >or=1, 0) and satisfaction (satisfied or very satisfied, 1; acceptable or unsatisfied, 0). Procedures of axillary blocks placement resulting in a comfort score of 3 and 2 were arbitrary considered as very comfortable and comfortable, respectively. Success rate of axillary blocks, time to perform block and complications related to procedures were noted. RESULTS: One hundred and twenty patients were included. In the ultrasound out-of-plane group, 55% (22/40) and 25% (10/40) of the procedures were very comfortable and comfortable as compared with 32% (13/40, P < 0.05) and 20% (8/40, P < 0.01) in the ultrasound in-plane group and 25% (10/40, P < 0.01) and 8% (3/40, P < 0.01) in the neurostimulation group, respectively. Duration of axillary placement was significantly smaller in the ultrasound out-of-plane group as compared with that of in-plane approaches (P < 0.05) and neurostimulation (P < 0.01). CONCLUSION: The present study showed that the ultrasound approaches were less painful and more comfortable than neurostimulation to place axillary blocks. We also showed that, although pain intensity resulting from blocks placement was similar with the ultrasound approaches, very comfortable procedures were more frequent with the out-of-plane than with the in-plane approach.

Spread of injectate associated with radial or median nerve-type motor response during infraclavicular brachial-plexus block: an ultrasound evaluation.

BACKGROUND AND OBJECTIVES: We have compared ultrasound characteristics of spread during infraclavicular brachial-plexus blocks by use of electrically evoked radial-nerve- or median-nerve-type distal motor responses to guide the injection of 30 mL of 1.5% mepivacaine. METHODS: Consecutive patients who required surgery distal to the upper arm were prospectively included in this study. With radial- or median-evoked distal motor response at a stimulating current intensity of less than 0.5 mA, patients were distributed into 2 equal groups. An independent investigator blinded to the evoked response described ultrasound characteristics of the spread of local anesthetic and assessed block quality 30 minutes after placement. A quality diffusion score proportional to the extent and intensity of spread around the axillary artery was used, and dynamic movements during injection were noted. RESULTS: Thirty-two patients were included. With radial-nerve-type motor response, the success rate of infraclavicular plexus block was 100%, but 3 supplemental axillary blocks were requested with median-nerve-type motor response. Quality diffusion scores were significantly higher with radial-nerve-type as compared with median-nerve-type motor response (P = .03). Injection after radial-nerve-type motor response resulted in a typical and reproducible ultrasound feature of posterior local-anesthetic spread associated with medial and upper movement of the axillary artery. With median-nerve-type motor response, failed blocks were associated with a specific posterior displacement of the axillary artery that resulted from superficial spread. CONCLUSION: We have demonstrated that as compared with median-nerve-type motor response, injection performed after a radial-nerve-type motor response promoted reproducible and remarkable ultrasound spread characteristics associated with complete sensory block of the 3 cords at 30 minutes.

Single-stimulation, low-volume infraclavicular plexus block: influence of the evoked distal motor response on success rate.

BACKGROUND AND OBJECTIVES: We compared the success rate of single-injection infraclavicular plexus block by using electrically evoked radial, ulnar, or median nerve-type distal motor response to guide the injection of local anesthetic. METHODS: Consecutive patients requiring surgery distal to the upper arm were prospectively included in this study over a 6-month period. No search for predetermined distal motor responses was performed. The first qualifying distal motor response evoked for a stimulating current intensity of <0.5 mA distributed patients into 3 groups of patients. The study was continued until 3 groups of 60 patients were fulfilled. Twenty to 25 minutes after the injection of 30 mL of 1.5% mepivacaine, blinded evaluation of block quality was performed. A successful block was defined by the presence of a complete sensory block of the 5 major nerve distal distributions of the arm. RESULTS: Five hundred patients were included. The first evoked distal motor response was of radial, median, and ulnar nerve type in 46% (n = 230), 42% (n = 210), and 12% (n = 60) cases, respectively. The success rate of the infraclavicular plexus block was significantly higher when the injection was performed on a radial nerve-type response (90%) as compared with the median (74%) or ulnar (68%) nerve distal motor response. Intraoperative sedation and general anesthesia were not needed. None of the patients experienced specific complications. CONCLUSION: We showed that evoked distal motor response influenced the success rate of single-injection infraclavicular plexus block. The highest success rate was obtained when injection was performed after radial nerve-type motor response.