Rabbit derived VL single-domains as promising scaffolds to generate antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.

Anacardium occidentale Bark as an Antidiabetic Agent.

Anacardium occidentale L. is used throughout the world to treat type 2 diabetes. In Portugal, a traditional herbal preparation made with stem bark of this species (AoBTHP) has been used for more than 30 years to treat this pathology. The AoBTHP was standardized on total phenolic content, and its hypoglycemic activity was assessed using db/db mice (n = 26) for 92 days. Three doses (40.2, 71.5, and 127.0 mg/kg/day, per os) were tested, and glibenclamide (5 mg/kg/day) was used as positive control. During the study, glycemia was measured under non-fasting or fasting states. In sequence, thin-layer chromatography bioautographic assays were used for the detection of possible alpha- and beta-glucosidase inhibitors. A significant hypoglycemic effect in fasting glycemia in days 31 and 57 was observed with the three tested doses. The 71.5 mg/kg and 127.0 mg/kg AoBTHPs significantly reduced non-fasting glycemia on day 24. The highest dose showed the most significant hypoglycemic effect. Gallic acid was identified as the major alpha- and beta-glucosidase inhibitor. The 127 mg/kg/day AoBTHP dose showed a greater glucose-lowering effect than glibenclamide. For the first time, a standardized AoBTHP was tested using an in vivo diabetes model, and its usage was preclinically validated for type 2 diabetes treatment. The hypoglycemic activity of an AoBTHP can be related to the presence of alpha- and beta-glucosidase inhibitors, such as gallic acid, but other mechanisms can also be involved.

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy.

Anti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identified six amino acid differences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived single-domain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.

A Prime-Boost Immunization Strategy with Vaccinia Virus Expressing Novel gp120 Envelope Glycoprotein from a CRF02_AG Isolate Elicits Cross-Clade Tier 2 HIV-1 Neutralizing Antibodies.

Development of new immunogens eliciting broadly neutralizing antibodies (bNAbs) is a main priority for the HIV-1 vaccine field. Envelope glycoproteins from non-B-non-C HIV-1clades have not been fully explored as components of a vaccine. We produced Vaccinia viruses expressing a truncated version of gp120 (gp120t) from HIV-1 clades CRF02_AG, H, J, B, and C and examined their immunogenicity in mice and rabbits. Mice primed with the recombinant Vaccinia viruses and boosted with the homologous gp120t or C2V3C3 polypeptides developed antibodies that bind potently to homologous and heterologous envelope glycoproteins. Notably, a subset of mice immunized with the CRF02_AG-based envelope immunogens developed a cross-reactive neutralizing response against tier 2 HIV-1 Env-pseudoviruses and primary isolates. Rabbits vaccinated with the CRF02_AG-based envelope immunogens also generated potent binding antibodies, and one animal elicited antibodies that neutralized almost all (13 of 16, 81.3%) tier 2 HIV-1 isolates tested. Overall, the results suggest that the novel CRF02_AG-based envelope immunogens and prime-boost immunization strategy elicit the type of immune responses required for a preventive HIV-1 vaccine.

Epidemiological Study of Pesticide Poisoning in Domestic Animals and Wildlife in Portugal: 2014-2020.

Nowadays the intentional poisoning of domestic and wild animals is a crime in the European Union (EU), but as in the past the poison is still used in rural areas of a number of European countries to kill animals that were considered harmful for human activities. From January 2014 up until October 2020, the Laboratory of Pharmacology and Toxicology of the Faculty of Veterinary Medicine (LFT-FMV) has done the analytical detection of poisoning substances in 503 samples of wildlife and domestic animals and pesticides residues were found in 239 of the samples analyzed. In this retrospective study, toxicology results from domestic species (dog, cat, sheep, cows, and horses), wildlife species (red foxes, birds of prey, lynx, and wild boar), and food baits, are presented. During this period the samples analyzed at the LFT-FMV, were received from all over the country. Analytical detections were performed via solvent extraction followed by thin layer chromatography. Molluscicides (47%, n = 109) and Carbamates (24%, n = 57) were found to be the first category of pesticides involved in intoxications, in both domestic and wild animals, followed by rodenticides (13%, n = 30)-in this group second and third generation, were the most represented; Strychnine is the third (11%, n = 26) even though this pesticide has been banned in Portugal since 1988 and in the European Union since 2006 and finally Organophosphates (5%, n = 11) in the small number. This study allowed to realize that a great number of positive samples involved banned pesticides (i.e., Aldicarb and Strychnine) but, at the same time, many positives cases were due to the exposure to commercially available products (i.e., Methiocarb and Anticoagulant rodenticides). Also, it's possible to identify the areas where domestic species are the most affected (i.e., Setubal and Lisboa) and the areas where the wild animals are the mainly affected species (i.e., Faro, Castelo Branco, and Bragança).

The histone deacetylase inhibitor panobinostat is a potent antitumor agent in canine diffuse large B-cell lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death in the United States and Europe. Although the outcome of NHL patients has improved over the last years with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic molecules and approaches. Histone deacetylase inhibitors (HDACis) have emerged as a powerful new class of anti-cancer drugs for human therapy. To investigate HDACi antitumor properties on canine diffuse large B-cell lymphoma, a panel of seven HDACi compounds (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) was screened on CLBL-1 canine B-cell lymphoma cell line. Our results demonstrated that all HDACis tested exhibited dose-dependent inhibitory effects on proliferation of CLBL-1 cells, while promoting increased H3 histone acetylation. Amongst all HDACis studied, panobinostat proved to be the most promising compound and was selected for further in vitro and in vivo evaluation. Panobinostat cytotoxicity was linked to H3 histone and α-tubulin acetylation, and to apoptosis induction. Importantly, panobinostat efficiently inhibited CLBL-1 xenograft tumor growth, and strongly induced acetylation of H3 histone and apoptosis in vivo. In conclusion, these results provide new data validating HDACis and, especially, panobinostat as a novel anti-cancer therapy for veterinary applications, while contributing to comparative oncology.

Preclinical randomized controlled trial of bilateral discectomy versus bilateral discopexy in Black Merino sheep temporomandibular joint: TEMPOJIMS - Phase 1- histologic, imaging and body weight results.

INTRODUCTION: The role of temporomandibular joint (TMJ) surgery is not well defined due to a lack of quality randomized controlled clinical trials, comparing different TMJ surgical treatments with medical and placebo interventions. The temporomandibular joint interposal study (TEMPOJIMS) is a rigorous preclinical trial divided in 2 phases. In phase 1 the authors investigated the role of the TMJ disc and in phase 2 the authors evaluated 3 different interposal materials. The present work of TEMPOJIMS - phase 1, aims to evaluate histopathologic and imaging changes of bilateral discectomy and discopexy in Black Merino sheep TMJ, using a high-quality trial following the ARRIVE guidelines. MATERIAL AND METHODS: This randomized, blinded and controlled preclinical trial was conducted in 9 Black Merino sheep to investigate histopathologic (primary outcome), imaging and body weight (secondary outcomes) changes after bilateral discectomy, discopexy and sham surgery. RESULTS: Significant changes were noticed in discectomy group, both in imaging and histopathologic analyses. Body weight changes were most pronounced in the discectomy group in the first 4 months after surgery with recovery to baseline weight 6 months after surgery. Discopexy induced nonsignificant changes in histopathologic, imaging and body weight analyses. CONCLUSIONS: This study reinforces the importance of developing an effective interposal material to substitute the TMJ disc and the need to explore the molecular mechanisms that underlie TMJ cartilage degeneration. The study design proposed in TEMPOJIMS represents an important progress towards future rigorous TMJ investigations.

Bioengineered Temporomandibular Joint Disk Implants: Study Protocol for a Two-Phase Exploratory Randomized Preclinical Pilot Trial in 18 Black Merino Sheep (TEMPOJIMS).

BACKGROUND: Preclinical trials are essential to test efficacious options to substitute the temporomandibular joint (TMJ) disk. The contemporary absence of an ideal treatment for patients with severe TMJ disorders can be related to difficulties concerning the appropriate study design to conduct preclinical trials in the TMJ field. These difficulties can be associated with the use of heterogeneous animal models, the use of the contralateral TMJ as control, the absence of rigorous randomized controlled preclinical trials with blinded outcomes assessors, and difficulties involving multidisciplinary teams. OBJECTIVE: This study aims to develop a new, reproducible, and effective study design for preclinical research in the TMJ domain, obtaining rigorous data related to (1) identify the impact of bilateral discectomy in black Merino sheep, (2) identify the impact of bilateral discopexy in black Merino sheep, and (3) identify the impact of three different bioengineering TMJ discs in black Merino sheep. METHODS: A two-phase exploratory randomized controlled preclinical trial with blinded outcomes is proposed. In the first phase, nine sheep are randomized into three different surgical bilateral procedures: bilateral discectomy, bilateral discopexy, and sham surgery. In the second phase, nine sheep are randomized to bilaterally test three different TMJ bioengineering disk implants. The primary outcome is the histological gradation of TMJ. Secondary outcomes are imaging changes, absolute masticatory time, ruminant time per cycle, ruminant kinetics, ruminant area, and sheep weight. RESULTS: Previous preclinical studies in this field have used the contralateral unoperated side as a control, different animal models ranging from mice to a canine model, with nonrandomized, nonblinded and uncontrolled study designs and limited outcomes measures. The main goal of this exploratory preclinical protocol is to set a new standard for future preclinical trials in oromaxillofacial surgery, particularly in the TMJ field, by proposing a rigorous design in black Merino sheep. The authors also intend to test the feasibility of pilot outcomes. The authors expect to increase the quality of further studies in this field and to progress in future treatment options for patients undergoing surgery for TMJ disk replacement. CONCLUSIONS: The study has commenced, but it is too early to provide results or conclusions.

Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model.

BACKGROUND: Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status. OBJECTIVES: To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance. METHODS: Healthy swine were randomized to receive by intracoronary route either 30x106 MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups. RESULTS: CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted. CONCLUSION: Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy.

Are vicilins another major class of legume lectins?

Legume lectins comprise a structurally related, Ca/Mn-dependent, widespread, abundant and well characterized lectin family when compared to the large number of lectins from other sources described in the literature. Strangely enough, no specific function has been assigned to them aside from a possible role in storage and/or defense. Using a recent and fine-tuned methodology capable of specific lectin identification, ß-conglutin, Vicia faba vicilin and ß-lathyrin, the vicilin storage globulins from Lupinus albus, V. faba and Lathyrus sativus, respectively, were shown to be capable of affinity binding to thoroughly washed erythrocyte membranes and of specific elution with appropriate sugars. Based on this evidence and on sparse data published in the literature, a second family of legume lectins is proposed: the 7S family of storage proteins from leguminous seeds, or family II of legume lectins. These lectins are also structurally related, widespread and well characterized. In addition, they self-aggregate in a Ca/Mg, electrostatic dependent manner and are even more abundant than the family I of legume lectins. Using the same evidence, reserve and defense roles may be attributed to family II of legume lectins.

Invasive assessment of the coronary microcirculation using the index of microcirculatory resistance: description and validation of an animal model.

INTRODUCTION: The index of microcirculatory resistance (IMR) enables/provides quantitative, invasive, and real-time assessment of coronary microcirculation status. AIMS: The primary aim of this study was to validate the assessment of IMR in a large animal model, and the secondary aim was to compare two doses of intracoronary papaverine, 5 and 10 mg, for induction of maximal hyperemia and its evolution over time. METHODS: Measurements of IMR were performed in eight pigs. Mean distal pressure (Pd) and mean transit time (Tmn) were measured at rest and at maximal hyperemia induced with intracoronary papaverine, 5 and 10 mg, and after 2, 5, 8 and 10 minutes. Disruption of the microcirculation was achieved by selective injection of 40-µm microspheres via a microcatheter in the left anterior descending artery. RESULTS: In each animal 14 IMR measurements were made. There were no differences between the two doses of papaverine regarding Pd response and IMR values - 11 ± 4.5 U with 5 mg and 10.6 ± 3 U with 10 mg (p=0.612). The evolution of IMR over time was also similar with the two doses, with significant differences from resting values disappearing after five minutes of intracoronary papaverine administration. IMR increased with disrupted microcirculation in all animals (41 ± 16 U, p=0.001). CONCLUSIONS: IMR provides invasive and real-time assessment of coronary microcirculation. Disruption of the microvascular bed is associated with a significant increase in IMR. A 5-mg dose of intracoronary papaverine is as effective as a 10-mg dose in inducing maximal hyperemia. After five minutes of papaverine administration there is no significant difference from resting hemodynamic status.

Maize and resistant starch enriched breads reduce postprandial glycemic responses in rats.

White wheat bread is a poor source of dietary fiber, typically containing less than 2%. A demand exists for the development of breads with starch that is slowly digestible or partially resistant to the digestive process. The utilization of maize flour and resistant starch is expected to reduce the release and absorption of glucose and, hence, lower the glycemic index of bread. This study was undertaken to investigate the hypothesis that a diet of maize bread, as produced and consumed in Portugal, would have beneficial metabolic effects on rats compared to white wheat bread. We also hypothesized that the effect of resistant starch on glycemic response could be altered by the use of different formulations and breadmaking processes for wheat and maize breads. Resistant starch (RS) was incorporated into formulations of breads at 20% of the inclusion rate of wheat and maize flours. Assays were conducted with male Wistar rats (n = 36), divided into four groups and fed either wheat bread, RS-wheat bread, maize bread, and RS-maize bread to evaluate feed intake, body weight gain, fecal pH, and postprandial blood glucose response (glycemic response). Blood triglycerides, total cholesterol concentrations, and liver weights were also determined. The maize bread group presented higher body weight gain and cholesterol level, lower fecal pH, and postprandial blood glucose response than the wheat bread group. The RS-wheat bread group showed significant reductions in feed intake, fecal pH, postprandial blood glucose response, and total cholesterol. The RS-maize group displayed significant reductions of body weight gain, fecal pH, and total cholesterol levels; however, for the glycemic response, only a reduction in fasting level was observed. These results suggest that maize bread has a lower glycemic index than wheat bread, and the magnitude of the effect of RS on glycemic response depends of type of bread.