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Immunomodulators are drugs that either stimulate or suppress the immune system in response to an immunopathological disease or cancer. The majority of clinically approved immunomodulators are either chemically synthesised (e.g., dexamethasone) or protein-based (e.g., monoclonal antibodies), whose uses are limited due to toxicity issues, poor bioavailability, or prohibitive cost. Nature is an excellent source of novel compounds, as it is estimated that almost half of all licenced medicines are derived from nature or inspired by natural product (NP) structures. The clinical success of the fungal-derived immunosuppressant cyclosporin A demonstrates the potential of natural products as immunomodulators. Conventionally, the screening of NP molecules for immunomodulation is performed in small animal models; however, there is a growing impetus to replace animal models with more ethical alternatives. One novel approach is the use of Galleria melonella larvae as an in vivo model of immunity. Despite lacking adaptive antigen-specific immunity, this insect possesses an innate immune system comparable to mammals. In this review, we will describe studies that have used this alternative in vivo model to assess the immunomodulating activity of synthetic and NP-derived compounds, outline the array of bioassays employed, and suggest strategies to enhance the use of this model in future research.

Background. Norovirus infection is the leading cause of acute nonbacterial gastroenteritis. Histoblood group antigens (HBGAs) are host susceptibility determinants for Norwalk virus (NV) infection. We hypothesized that antibodies that block NV-HBGA binding are associated with protection from clinical illness following NV exposure. Methods. We developed an HBGA blocking assay to examine the ability of human serum to block the interaction of NV viruslike particles with H type 1 and H type 3 glycans. Serum samples from persons who were experimentally challenged with NV were evaluated. Results. There was a high correlation between the H type 1 and H type 3 synthetic glycan assays (r=0.977; P<.001); the H type 1 assay had higher quantitative sensitivity (P<.001). Among 18 infected secretor-positive individuals, blocking titers peaked by day 28 after challenge and were higher for individuals who did not develop gastroenteritis than for those who developed gastroenteritis on days 0, 14, 28, and 180 (P<.05 for each). In addition, 6 of 6 subjects without gastroenteritis had measurable prechallenge blocking titers (>25), compared with 2 of 12 subjects with gastroenteritis (P=.002). Conclusions. Blocking antibodies correlate with protection against clinical NV gastroenteritis. This knowledge will help guide the evaluation of new vaccine strategies and the elucidation of the nature of immunity to the virus. Trial registration. ClinicalTrials.gov identifier: NCT00138476.

Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) antibodies have provided the binding element of the majority of these assays but recombinant antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins. Graphical Abstract Schematic representation of (A) affinity selection of phage-displayed recombinant antibody (rAb) and (B) immunoglobulin antibody structures and the corresponding antibody fragments

There is growing recognition that animal methods bias, a preference for animal‐based methods where they are not necessary or where nonanimal‐based methods may already be suitable, can impact the likelihood or timeliness of a manuscript being accepted for publication. Following April 2022 workshop about animal methods bias in scientific publishing, a coalition of scientists and advocates formed a Coalition to Illuminate and Address Animal Methods Bias (COLAAB). The COLAAB has developed this guide to be used by authors who use nonanimal methods to avoid and respond to animal methods bias from manuscript reviewers. It contains information that researchers may use during 1) study design, including how to find and select appropriate nonanimal methods and preregister a research plan, 2) manuscript preparation and submission, including tips for discussing methods and choosing journals and reviewers that may be more receptive to nonanimal methods, and 3) the peer review process, providing suggested language and literature to aid authors in responding to biased reviews. The author's guide for addressing animal methods bias in publishing is a living resource also available online at animalmethodsbias.org, which aims to help ensure fair dissemination of research that uses nonanimal methods and prevent unnecessary experiments on animals.

Background The overuse of antibiotics has led to increased antimicrobial resistance, but plant-derived biological response modifiers represent a potential alternative to these drugs. This investigation examined the immunomodulatory and antibacterial activities of Sida cordifolia (used in ethnomedicinal systems to treat infectious disease). Methods Successive extractions were performed from the roots of these plants in hexane, chloroform, methanol and water. Immunomodulatory activity was determined in a series of experiments measuring the responses of splenocytes, macrophages and an in vivo model of innate immunity ( Galleria mellonella ) . Antibacterial activity was assessed by determining minimum inhibitory/bactericidal concentrations (MIC/MBCs) for various Gram-positive and Gram-negative bacterial strains. Results Immunomodulatory activity was confined to the aqueous extract, and further fractionation and biochemical analysis yielded a highly potent polysaccharide-enriched fraction (SCAF5). SCAF5 is a complex mixture of different polysaccharides with multiple immunomodulatory effects including immune cell proliferation, antibody secretion, phagocytosis, nitric oxide production, and increased expression of pro-inflammatory cytokines. Furthermore, Galleria mellonella pre-treated with SCAF5 produced more haemocytes and were more resistant ( P  < 0.001) to infection with methicillin-resistant Staphylococcus aureus (MRSA) with a 98% reduction in bacterial load in pre-treated larvae compared to the negative control. The antibacterial activity of Sida cordifolia was confined to the methanolic fraction. Extensive fractionation identified two compounds, rosmarinic acid and its 4-O-β-d-glucoside derivative, which had potent activity against Gram-positive antibiotic-resistant bacteria, including MRSA. Conclusions Sida cordifolia counters bacterial infections through a dual mechanism, and immunomodulatory polysaccharides from this plant should be isolated and characterised to realise their potential as anti-infective agents. Such properties could be developed as an antibiotic alternative (1) in the clinic and (2) alternative growth promoter for the agri-food industry.

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

The immunogenicity of proteins encapsulated in poly( dl-lactide-co-glycolide) (PLG) microspheres has not been investigated to any extent in large animal models. In this study, IgG and IgA responses to ovalbumin (OVA), encapsulated in microspheres was investigated following intranasal inoculation into calves. Scanning electron microscopy and flow cytometric analysis demonstrated a uniform microsphere population with a diameter of <2.5 μm. Ovalbumin was released steadily from particles stored in PBS almost in a linear fashion, and after 4 weeks many particles showed cracks and fissures in their surface structure. Following intranasal inoculation of calves with different doses of encapsulated antigen, mean levels of ovalbumin-specific IgA were observed to increase steadily but significant differences in IgA levels (from the pre-inoculation level) were only observed following a second intranasal inoculation. With 0.5 and 1.0 mg doses of antigen, ovalbumin-specific IgG was also detected in serum. Ovalbumin-specific IgA persisted in nasal secretions for a considerable period of time and were still detectable in four out of seven animals, 6 months after inoculation.

After two decades of frustration, genetic studies of schizophrenia have entered an era of spectacular success. Advances in genotyping technologies and high throughput sequencing, increasing analytic rigour and collaborative efforts on a global scale have generated a profusion of new findings. The broad conclusions from these studies are threefold: (1) schizophrenia is a highly polygenic disorder with a complex array of contributing risk loci across the allelic frequency spectrum; (2) many psychiatric illnesses share risk genes and alleles, specifically, schizophrenia has substantial overlaps with bipolar disorder, intellectual disability, major depressive disorder and autism spectrum disorders; and (3) some convergent biological themes are emerging from studies of schizophrenia and related disorders. In this commentary, we focus on the very recent findings that have emerged in the past 12 months, and in particular, the areas of convergence that are beginning to emerge from multiple study designs.

Inherited alleles account for most of the genetic risk for schizophrenia. However, new ( de novo ) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N -methyl- d -aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case–control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders. The authors report the largest family-trio exome sequencing study of schizophrenia to date; mutations are overrepresented in genes for glutamatergic synaptic proteins and also genes mutated in autism and intellectual disability, providing insights into aetiological mechanisms and pathopshyisology shared with other neurodevelopmental disorders. Pathogenic mechanisms in schizophrenia Two major sequencing studies of the exome — the protein-coding portion of the genome — in schizophrenia sufferers and their relatives are published in this issue of Nature . Together they provide strong pointers to specific pathogenic mechanisms that disrupt the glutamatergic synapses in schizophrenia. In particular, mutations that influence the action of the scaffold protein ARC (activity-regulated cytoskeleton-associated protein) are prominently involved, as are mutations in targets of the fragile X mental retardation protein (FMRP). Defects in FMRP have previously been shown to be associated with autism spectrum disorders.