Mitigating animal methods bias to reduce animal use and improve biomedical translation.

Nonanimal biomedical research methods have advanced rapidly over the last decade making them the first-choice model for many researchers due to improved translatability and avoidance of ethical concerns. Yet confidence in novel nonanimal methods is still being established and they remain a small portion of nonclinical biomedical research, which can lead peer reviewers to evaluate animal-free studies or grant proposals in a biased manner. This "animal methods bias" is the preference for animal-based research methods where they are not necessary or where nonanimal-based methods are suitable. It affects the fair consideration of animal-free biomedical research, hampering the uptake and dissemination of these approaches by putting pressure on researchers to conduct animal experiments and potentially perpetuating the use of poorly translatable model systems. An international team of researchers and advocates called the Coalition to Illuminate and Address Animal Methods Bias (COLAAB) aims to provide concrete evidence of the existence and consequences of this bias and to develop and implement solutions towards overcoming it. The COLAAB recently developed the first of several mitigation tools: the Author Guide for Addressing Animal Methods Bias in Publishing, which is described herein along with broader implications and future directions of this work.

Author Guide for Addressing Animal Methods Bias in Publishing.

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.

Proceedings of a workshop to address animal methods bias in scientific publishing.

Animal methods bias in scientific publishing is a newly defined type of publishing bias describing a preference for animal-based methods where they may not be necessary or where nonanimal-based methods may already be suitable, which impacts the likelihood or timeliness of a manuscript being accepted for publication. This article covers the output from a workshop between stakeholders in publishing, academia, industry, government, and non-governmental organizations. The intent of the workshop was to exchange perspectives on the prevalence, causes, and impact of animal methods bias in scientific publishing, as well as to explore mitigation strategies. Output from the workshop includes summaries of presentations, breakout group discussions, participant polling results, and a synthesis of recommendations for mitigation. Overall, participants felt that animal methods bias has a meaningful impact on scientific publishing, though more evidence is needed to demonstrate its prevalence. Significant consequences of this bias that were identified include the unnecessary use of animals in scientific procedures, the continued reliance on animals in research ­ even where suitable nonanimal methods exist, poor rates of clinical translation, delays in publication, and negative impacts on career trajectories in science. Workshop participants offered recommendations for journals, publishers, funders, governments, and other policy makers, as well as the scientific community at large, to reduce the prevalence and impacts of animal methods bias. The workshop resulted in the creation of working groups committed to addressing animal methods bias, and activities are ongoing.

Acute aerobic exercise-conditioned serum reduces colon cancer cell proliferation in vitro through interleukin-6-induced regulation of DNA damage.

Epidemiological evidence shows that regular physical activity is associated with reduced risk of primary and recurrent colon cancer. However, the underlying mechanisms of action are poorly understood. We evaluated the effects of stimulating a human colon cancer cell line (LoVo) with human serum collected before and after an acute exercise bout vs nonexercise control serum on cancer cell proliferation. We also measured exercise-induced changes in serum cytokines and intracellular protein expression to explore potential biological mechanisms. Blood samples were collected from 16 men with lifestyle risk factors for colon cancer (age ≥50 years; body mass index ≥25 kg/m2 ; physically inactive) before and immediately after an acute bout of moderate-intensity aerobic interval exercise (6 × 5 minutes intervals at 60% heart rate reserve) and a nonexercise control condition. Stimulating LoVo cells with serum obtained immediately after exercise reduced cancer cell proliferation compared to control (-5.7%; P = .002). This was accompanied by a decrease in LoVo cell γ-H2AX expression (-24.6%; P = .029), indicating a reduction in DNA damage. Acute exercise also increased serum IL-6 (24.6%, P = .002). Furthermore, stimulating LoVo cells with recombinant IL-6 reduced γ-H2AX expression (ß = -22.7%; P < .001) and cell proliferation (ß = -5.3%; P < .001) in a linear dose-dependent manner, mimicking the effect of exercise. These findings suggest that the systemic responses to acute aerobic exercise inhibit colon cancer cell proliferation in vitro, and this may be driven by IL-6-induced regulation of DNA damage and repair. This mechanism of action may partly underlie epidemiological associations linking regular physical activity with reduced colon cancer risk.

Extracts of Sida cordifolia contain polysaccharides possessing immunomodulatory activity and rosmarinic acid compounds with antibacterial activity.

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-ß-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.

Global prevalence of burnout among postgraduate medical trainees: a systematic review and meta-regression.

BACKGROUND: Burnout among postgraduate medical trainees (PMTs) is increasingly being recognized as a crisis in the medical profession. We aimed to establish the prevalence of burnout among PMTs, identify risk and protective factors, and assess whether burnout varied by country of training, year of study and specialty of practice. METHODS: We systematically searched MEDLINE, Embase, PsycINFO, the Cochrane Database of Systematic Reviews, Web of Science and Education Resources Information Center from their inception to Aug. 21, 2018, for studies of burnout among PMTs. The primary objective was to identify the global prevalence of burnout among PMTs. Our secondary objective was to evaluate the association between burnout and country of training, year of study, specialty of training and other sociodemographic factors commonly thought to be related to burnout. We employed random-effects meta-analysis and meta-regression techniques to estimate a pooled prevalence and conduct secondary analyses. RESULTS: In total, 8505 published studies were screened, 196 met eligibility and 114 were included in the meta-analysis. The pooled prevalence of burnout was 47.3% (95% confidence interval 43.1% to 51.5%), based on studies published over 20 years involving 31 210 PMTs from 47 countries. The prevalence of burnout remained unchanged over the past 2 decades. Burnout varied by region, with PMTs of European countries experiencing the lowest level. Burnout rates among medical and surgical PMTs were similar. INTERPRETATION: Current wellness efforts and policies have not changed the prevalence of burnout worldwide. Future research should focus on understanding systemic factors and leveraging these findings to design interventions to combat burnout. STUDY REGISTRATION: PROSPERO no. CRD42018108774.

Surface plasmon resonance biosensor for the determination of 3-methyl-quinoxaline-2-carboxylic acid, the marker residue of olaquindox, in swine tissues.

To monitor the illegal use of olaquindox in animals, a monoclonal antibody-based surface plasmon resonance (SPR) biosensor method has been developed to detect 3-methyl-quinoxaline-2-carboxylic acid, the marker residues of olaquindox, in swine tissues. The limit of detection was 1.4 µg kg-1 in swine muscle and 2.7 µg kg-1 in swine liver, which are lower than the EU recommended concentration (10 µg kg-1). The recoveries were from 82% to 104.6%, with coefficients of variation of less than 12.2%. Good correlations between SPR and HPLC results (r = 0.9806, muscle; r = 0.9698, liver) and between SPR and ic-ELISA results (r = 0.9918, muscle; r = 0.9873, liver) were observed in the affected tissues, which demonstrated the reliability of the SPR method. This method would be a rapid and reliable tool for the screening of the residues of olaquindox in the edible tissues of animals.

Digital Rectal Examination for Prostate Cancer Screening in Primary Care: A Systematic Review and Meta-Analysis.

PURPOSE: Although the digital rectal examination (DRE) is commonly performed to screen for prostate cancer, there is limited data to support its use in primary care. This review and meta-analysis aims to evaluate the diagnostic accuracy of DRE in screening for prostate cancer in primary care settings. METHODS: We searched MEDLINE, Embase, DARE (Database of Abstracts of Reviews of Effects), Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and CINAHL (Cumulative Index to Nursing and Allied Health Literature) from their inception to June 2016. Six reviewers, in pairs, independently screened citations for eligibility and extracted data. Pooled estimates were calculated for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of DRE in primary care settings using an inverse-variance meta-analysis. We used QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies 2) and GRADE (Grades of Recommendation Assessment, Development, and Evaluation) guidelines to assess study risk of bias and quality. RESULTS: Our search yielded 8,217 studies, of which 7 studies with 9,241 patients were included after the screening process. All patients analyzed underwent both DRE and biopsy. Pooled sensitivity of DRE performed by primary care clinicians was 0.51 (95% CI, 0.36-0.67; I2 = 98.4%) and pooled specificity was 0.59 (95% CI, 0.41-0.76; I2 = 99.4%). Pooled PPV was 0.41 (95% CI, 0.31-0.52; I2 = 97.2%), and pooled NPV was 0.64 (95% CI, 0.58-0.70; I2 = 95.0%). The quality of evidence as assessed with GRADE was very low. CONCLUSION: Given the considerable lack of evidence supporting its efficacy, we recommend against routine performance of DRE to screen for prostate cancer in the primary care setting.

Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins.

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.

Seroepidemiology of norovirus-associated travelers' diarrhea.

BACKGROUND: Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers' diarrhea (TD) in US and European travelers to Mexico, Guatemala, and India. METHODS: Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using reverse transcription-polymerase chain reaction (RT-PCR). Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLPs) to measure serum levels of immunoglobulin A (IgA) and IgG by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA); serum IgM was measured by capture enzyme-linked immunosorbent assay (ELISA), and the 50% antibody-blocking titer (BT50 ) was determined by a carbohydrate-blocking assay. RESULTS: NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or fourfold or more rise in antibody titer. Significantly more individuals had detectable preexisting IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p = 0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p = 0.0037), IgG (p = 0.003), and BT50 (p = <0.0001). None of the NoV-infected TD travelers had BT50 > 200, a level that has been described previously as a possible correlate of protection. CONCLUSIONS: We found that GI-NoVs are commonly associated with TD cases identified in US adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.

Immunogenetic responses in calves to intranasal delivery of bovine respiratory syncytial virus (BRSV) epitopes encapsulated in poly (DL-lactide-co-glycolide) microparticles.

Bovine respiratory syncytial virus (BRSV) is the principal aetiological agent of the bovine respiratory disease complex. A BRSV subunit vaccine candidate consisting of two synthetic peptides representing putative protective epitopes on BRSV surface glycoproteins in soluble form or encapsulated in poly(lactide-co-glycolide) (PLG) microparticles were prepared. Calves (10 weeks old) with diminishing levels of BRSV-specific maternal antibody were intranasally administered a single dose of the different peptide formulations. Peptide-specific local immune responses (nasal secretion IgA), but not systemic humoral (serum IgG) or cellular responses (serum IFN-γ), were generated by all forms of peptide. There was a significant reduction in occurrence of respiratory disease in the animals inoculated with all peptide formulations compared to animals given PBS alone. Furthermore no adverse effects were observed in any of the animals post vaccination. These results suggest that intranasal immunisation with the peptide subunit vaccine does induce an as yet unidentified protective immune response.

Local and systemic immune responses in mice to intranasal delivery of peptides representing bovine respiratory syncytial virus epitopes encapsulated in poly (DL-lactide-co-glycolide) microparticles.

The potential of a microparticulate vaccine delivery system in eliciting a specific mucosal antibody response in the respiratory tract of mice was evaluated. Two vaccine candidate peptides representing epitopes from the G attachment and F fusion antigens from bovine respiratory syncytial virus (BRSV) were encapsulated into poly(DL-lactide co-glycolide) biodegradable microparticles. The encapsulation process did not denature the entrapped peptides as verified by detection of peptide-specific antibodies in mucosal secretions by ELISA using peptide as antigen. Following intranasal immunisation, the encapsulated peptides induced stronger upper and lower respiratory tract specific-IgA responses, respectively, than the soluble peptide forms. Moreover, a strong peptide-specific cell-mediated immune response was measured in splenocytes in vitro from the mice inoculated with the encapsulated peptides compared to their soluble form alone indicating that migration of primed T cells had taken place from the site of mucosal stimulation in the upper respiratory tract to the spleen. These results act as a foundation for vaccine efficacy studies in large animal BRSV challenge models.

A time-resolved immunoassay to measure serum antibodies to the rotavirus VP6 capsid protein.

The rotavirus (RV) inner capsid protein VP6 is widely used to evaluate immune response during natural infection and in vaccine studies. Recombinant VP6 from the most prevalent circulating rotavirus strains in each subgroup (SG) identified in a birth cohort of children in southern India [SGII (G1P[8]) and SGI (G10P[11])] were produced. The purified proteins were used to measure VP6-specific antibodies in a Dissociation-Enhanced Lanthanide Fluorometric Immunoassay (DELFIA). The ability of the assay to detect a ≥2 fold rise in IgG level in a panel of serum samples from a longitudinal study was compared to a gold standard virus-capture ELISA. A strong association was observed between the assays (p<0.001; chi-squared test) with assay performances remaining similar when the samples were subdivided as having a fold change increase in VP6 antibody levels (a) within 90 days of RV RNA detection in stool or (b) if no RV RNA was detected within that time period. This study demonstrates the suitability of using recombinant proteins to measure anti-RV immune responses and serves as a "proof of principle" to examine the antibody responses generated to other recombinant RV proteins and thereby possibly identify a correlate of protection.

Serological responses to experimental Norwalk virus infection measured using a quantitative duplex time-resolved fluorescence immunoassay.

A quantitative duplex time-resolved fluorescence assay, dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), was developed to measure Norwalk virus (NV)-specific IgA and IgG antibodies simultaneously. The duplex assay showed superior performance by detecting seroconversion following experimental NV infection at an earlier time point than a reference total immunoglobulin enzyme-linked immunosorbent assay (ELISA).

Adsorption and aggregation properties of norovirus GI and GII virus-like particles demonstrate differing responses to solution chemistry.

The transport properties (adsorption and aggregation behavior) of virus-like particles (VLPs) of two strains of norovirus ("Norwalk" GI.1 and "Houston" GII.4) were studied in a variety of solution chemistries. GI.1 and GII.4 VLPs were found to be stable against aggregation at pH 4.0-8.0. At pH 9.0, GI.1 VLPs rapidly disintegrated. The attachment efficiencies (α) of GI.1 and GII.4 VLPs to silica increased with increasing ionic strength in NaCl solutions at pH 8.0. The attachment efficiency of GI.1 VLPs decreased as pH was increased above the isoelectric point (pH 5.0), whereas at and below the isoelectric point, the attachment efficiency was erratic. Ca(2+) and Mg(2+) dramatically increased the attachment efficiencies of GI.1 and GII.4 VLPs, which may be due to specific interactions with the VLP capsids. Bicarbonate decreased attachment efficiencies for both GI.1 and GII.4 VLPs, whereas phosphate decreased the attachment efficiency of GI.1, while increasing GII.4 attachment efficiency. The observed differences in GI.1 and GII.4 VLP attachment efficiencies in response to solution chemistry may be attributed to differential responses of the unique arrangement of exposed amino acid residues on the capsid surface of each VLP strain.

Serological correlate of protection against norovirus-induced gastroenteritis.

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.

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties.

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.

Antigen-specific IgA and IgG responses in calves inoculated intranasally with ovalbumin encapsulated in poly(DL-lactide-co-glycolide) microspheres.

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 micrometers. 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 1.0mg 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.