Social play in African savannah elephants may inform selection against aggression.

In social groups, competition for individual advantage is balanced with cooperation, for the collective benefit. Selection against aggression has favored cooperation and non-aggressive competitive strategies. Because social play is a behavioral system that fluctuates between cooperation and competition, selection against aggression might have especially influenced this behavior.

Heavy chain-only antibodies with a stabilized human VH in transgenic chickens for therapeutic antibody discovery.

Heavy chain-only antibodies have found many applications where conventional heavy-light heterodimeric antibodies are not favorable. Heavy chain-only antibodies with their single antigen-binding domain offer the advantage of a smaller size and higher stability relative to conventional antibodies, and thus, the potential for novel targeting modalities. Domain antibodies have commonly been sourced from camelids with humanization or transgenic rodents expressing heavy chains without light chains, but these host species are all mammalian, limiting their capacity to elicit robust immune responses to conserved mammalian targets.

Witness for resolution: post-conflict quadratic affiliation in semi-free ranging pigs.

In social mammals, post-conflict resolution can involve the reunion of former opponents (reconciliation), spontaneous/solicited post-conflict affiliation of a third party with either opponent (triadic contacts), and affiliation between other individuals (hereafter bystanders; quadratic contacts). Quadratic contacts-possibly informing complex cognitive abilities-have been neglected in post-conflict studies. We investigated quadratic affiliation in semi-free ranging pigs , at the ethical farm Parva-Domus (Cavagnolo, Italy).

Chickens with a Truncated Light Chain Transgene Express Single-Domain H Chain-Only Antibodies.

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system.

Sows' Responses to Piglets in Distress: An Experimental Investigation in a Natural Setting.

Domestic pigs () possess complex socio-cognitive skills, and sows show high inter-individual variability in maternal behaviour. To evaluate how females-reared under natural conditions-react to the isolation calls of their own piglets or those of other females, we conducted observations and experimental trials. In January-February 2021, we conducted all-occurrences sampling on affiliation, aggression, and lactation (daily, 7:30-16:30 h) on six lactating and four non-lactating females at the ethical farm Parva Domus (Turin, Italy).

Play fighting versus real fighting in piglets (Sus scrofa): Similar patterns, different structure.

When animals engage in 'non-serious' fighting (play-fighting) they 'borrow' motor patterns especially from the aggressive context. It may be difficult to distinguish play- and real-fighting. This is particularly true for piglets (Sus scrofa), which can use play-fighting as a substitute for aggression.

Domestic pigs (Sus scrofa) engage in non-random post-conflict affiliation with third parties: cognitive and functional implications.

In social mammals, conflict resolution involves the reunion of former opponents (aggressor and victim) after an aggressive event (reconciliation) or post-conflict triadic contacts with a third party, started by either opponent (solicited-TSC) or spontaneously offered by the third party (unsolicited-TUC). These post-conflict strategies can serve different functions, including consolation (specifically when TUCs reduce the victim's anxiety). We investigated the possible presence and modulating factors of such strategies on semi-free ranging pigs (Sus scrofa; N = 104), housed at the ethical farm Parva Domus (Cavagnolo, Italy).

Does the Domestication Syndrome Apply to the Domestic Pig? Not Completely.

The 'domestication syndrome' defines a suite of features that domesticated animals possess as the result of the artificial selection operated by since the Neolithic. An interesting anthropological question is whether such features, including increased tameness and reduced aggression, apply to all domesticated forms. We investigated this issue in the domestic pig ().

Anxiety Behavior in Pigs () Decreases Through Affiliation and May Anticipate Threat.

Anxiety is a physio-psychological state anticipating an imminent threat. In social mammals it is behaviorally expressed displacement activities and buffered affiliation. Anxiety research on domestic pigs () has mostly focused on abnormal/stereotypic behavior associated with intensive farming.

Production of Transgenic Chickens Using Cultured Primordial Germ Cells and Gonocytes.

The unique characteristics of the avian embryo, with its large opaque yolk, have necessitated the development of different approaches to transgenesis from those that have been successful in mammalian species. Genetic modification of birds was greatly advanced by the ability to grow long-term cultures of primordial germ cells (PGCs). These cells are obtained from embryos, established in culture, and can be propagated without losing the ability to contribute to the germline when reintroduced into a host animal.

A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3.

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.

Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets.

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans.

Expression of heavy chain-only antibodies can support B-cell development in light chain knockout chickens.

Since the discovery of antibody-producing B cells in chickens six decades ago, chickens have been a model for B-cell development in gut-associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL(-/-) ) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery.

High-efficiency antibody discovery achieved with multiplexed microscopy.

The analysis of secreted antibody from large and diverse populations of B cells in parallel at the clonal level can reveal desirable antibodies for diagnostic or therapeutic applications. By immobilizing B cells in microdroplets with particulate reporters, decoding and isolating them in a microscopy environment, we have recovered panels of antibodies with rare attributes to therapeutically relevant targets. The ability to screen up to 100 million cells in a single experiment can be fully leveraged by accessing primary B-cell populations from evolutionarily divergent species such as chickens.

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny.

Generation of chickens expressing Cre recombinase.

Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with β-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated.

Inserting random and site-specific changes into the genome of chickens.

During the past decade, modifications to the chicken genome have evolved from random insertions of small transgenes using viral vectors to site-specific deletions using homologous recombination vectors and nontargeted insertions of large transgenes using phi-31 integrase. Primordial germ cells (PGC) and gonocytes are the germline-competent cell lines in which targeted modifications and large transgenes are inserted into the genome. After extended periods of in vitro culture, PGC retain their capacity to form functional gametes when reintroduced in vivo.

Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells.

Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species.

Harnessing gene conversion in chicken B cells to create a human antibody sequence repertoire.

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion.

Interspecific germline transmission of cultured primordial germ cells.

In birds, the primordial germ cell (PGC) lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis.

Potent high-affinity antibodies for treatment and prophylaxis of respiratory syncytial virus derived from B cells of infected patients.

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million.

Antibody discovery via multiplexed single cell characterization.

The secreted immunoglobulin footprint of single hybridoma cells, containing ~10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell's product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen.

Multiplexed Elispot assay.

Micron scale latex beads are well established as highly biocompatible reagents. Imbibing two fluorescent dyes into the interior of the beads enables the creation of a family of combinatorially colored labels. Previous use of such beads, in flow cytometry for example, has focused on beads of approximately 5 microm diameter.

A novel method for depositing erythroid cells onto glass slides for fetal cell analysis.

Background: We have developed a method for selecting erythroblasts from blood, the first step toward identifying fetal cells in maternal blood for diagnostic purposes. Because the selection method results in a large number of positive cells, we needed to develop new methods to deposit the cells onto slides and to modify in situ hybridization procedures to enable detection of fetal cells.

Methods: We utilized Nunc flaskettes to increase the slide surface area available for cell deposition.

Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood.

Background: Many methods have been employed to obtain fetal cells from maternal blood for prenatal diagnostics, but there has been little work done that compares the efficacy of different methods. This study presents a comparison of two commonly used methods for selecting erythroblasts with selection directly from whole blood.

Methods: Erythroblasts were isolated from maternal blood by either differential lysis or density separation, followed by selection with an antibody to the transferrin receptor.