A Cross-sectional Survey on Rodent Environmental Health Monitoring Practices: Benchmarking, Associations, and Barriers.

Tens of thousands of rodents are used each year in Rodent Health Monitoring programs. However, Environment Health Monitoring (EHM) could replace sentinel rodent use while maintaining or even improving diagnostic quality. Despite its advantages, widespread implementation of EHM appears to be relatively low. To better understand EHM's prevalence and factors influencing its use, we surveyed research animal professionals. Our hypotheses were (1) EHM prevalence would be low and (2) EHM use would be associated with beliefs and knowledge about EHM. Participants were recruited via online promotion. A total of 158 individuals completed a mixed-methods survey about current practices, beliefs, and knowledge about EHM. Qualitative data were coded using thematic analysis and analyzed using generalized linear models. Results showed that current EHM implementation was low; only 11% of institutions used EHM exclusively. Across the 111 institutions surveyed, over 20,000 soiled bedding sentinels were used each year. However, most participants believed EHM to be advantageous in replacing sentinel animals (78% of participants). Some participants believed EHM could save time (31%), cost less (27%), and be highly accurate (15%). Conversely, some participants believed EHM would be difficult to use due to their current caging type (40%), higher costs (21%), lower accuracy (16%), and personnel attitudes/expertise (14%). Overall, respondents with higher planned EHM use also had more positive attitudes, norms, and control of EHM. We also identified several factors that could promote the implementation of EHM. Communication efforts should emphasize that EHM is compatible with various types of caging, can provide cost savings, has high accuracy, and is consistent with the 3Rs as a replacement. Efforts should also focus on improving attitudes, encouraging peers, and providing resources to facilitate implementation. Implementation in just the surveyed institutions could eliminate the need for well over 20,000 rodents each year, consistent with 3Rs goals.

A scalable and robust cationic lipid/polymer hybrid nanoparticle platform for mRNA delivery.

mRNA based gene therapies hold the potential to treat multiple diseases with significant advantages over DNA based therapies, including rapid protein expression and minimized risk of mutagenesis. However, successful delivery of mRNA remains challenging, and clinical translation of mRNA therapeutics has been limited. This study investigated the use of a lipid/polymer hybrid (LPH) nanocarrier for mRNA, designed to address key delivery challenges and shuttle mRNA to targeted tissues. LPH nanocarriers were synthesized using a scalable microfluidic process with a variety of material compositions and mRNA loading strategies. Results show that a combination of permanently ionized and transiently, pH-dependent ionizable cationic lipids had a synergistic effect upon on mRNA gene translation, when compared to each lipid independently. Upon intravenous administration, particles with adsorbed mRNA outperformed particles with encapsulated mRNA for protein expression in the lungs and the spleen despite significant LPH nanoparticle localization to the liver. In contrast, encapsulated particles had higher localized expression when injected intramuscularly with protein expression detectable out to 12 days post injection. Intramuscular administration of particles with OVA mRNA resulted in robust humoral immune response with encapsulated outperforming adsorbed particles in terms of antibody titers at 28 days. These results demonstrate LPH nanocarriers have great potential as a vehicle for mRNA delivery and expression in tissues and that tissue expression and longevity can be influenced by LPH composition and route of administration.

Risk Assessment for Use of a Porcine Circovirus-Contaminated Reagent in a Barrier Maintained Rodent Colony.

A proposal for the use of porcine pancreatic elastase (PPE) to develop a mouse model of pulmonary emphysema raised concerns about introducing contaminating porcine viruses into our barrier facility. Porcine Circovirus (PCV) is a known contaminant of vaccines and cell cultures that have been exposed to porcine-derived reagents. Endemic infection of PCV3 in laboratory mice has been reported, and some evidence supports natural PCV infection in wild mice. PPE samples from 2 different vendors tested positive for DNA from both PCV2 and 3. To allow model development with these reagents to proceed, we developed a protocol that would meet scientific objectives, minimize exposure of mice, and provide information on the potential for the virus to spread. Five d after BALB/c mice received intralaryngeal administration of PPE, lungs were harvested and analyzed for evidence of disease. Tissues from other major organs were submitted to test for disseminated PCV2 and 3 DNA. Similarly, tissues (including lungs) from direct contact nude sentinel mice were analyzed for the presence of the virus. To evaluate the possibility of endemic PCV2/3 infection, we also surveyed non-porcine reagent exposed mice on other studies. PCV2 and 3 was not detected in any of the tissues submitted. Although this study provided no evidence of infection and transmission of PCV2/3 from the contaminated PPE sample over the 5 d study, further work is needed to understand the risks and impact of introducing PCV contaminated cells or reagents into barrier maintained rodent colonies.

Application of microphysiological systems in biopharmaceutical research and development.

Within the last 10 years, several tissue microphysiological systems (MPS) have been developed and characterized for retention of morphologic characteristics and specific gene/protein expression profiles from their natural in vivo state. Once developed, their utility is typically further tested by comparing responses to known toxic small-molecule pharmaceuticals in efforts to develop strategies for further toxicity testing of compounds under development. More recently, application of this technology in biopharmaceutical (large molecules) development is beginning to be more appreciated. In this review, we describe some of the advances made for tissue-specific MPS and outline the advantages and challenges of applying and further developing MPS technology in preclinical biopharmaceutical research.

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis.

Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.

Tumor uptake of pegylated diabodies: Balancing systemic clearance and vascular transport.

The accumulation, dissemination and clearance of monoclonal antibody-based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)-modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG-modified diabodies improved the PK of the parental diabody from a half-life of 40 min to over 40 h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody-PEG5K showing peak accumulation early on, but with the larger diabody-PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody-PEG20K-B with a hydrodynamic radius (Rh) of 6 nm had superior tumor uptake than the larger diabody-PEG40K-B with Rh of 12 nm, indicating that beyond 6 nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ~6 nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.

To Treat or Not to Treat: The Effects of Pain on Experimental Parameters.

A common dilemma faced by all animal bioethics committees arises when exceptions are proposed to the use of analgesics in painful procedures. The committee and researcher must weigh the possible confounding effects of including additional drugs (analgesics) in their treatment regimen against the moral obligation to perform humane research. Often neglected in these considerations are the potential confounding effects of unrelieved pain and consistency with pain-relieving practices in human medicine. In this review, we summarize what is currently known regarding the molecular and physiologic effects of pain and analgesics in common animal models used across several therapeutic areas. This work is intended to help provide guidance and assurance that a comprehensive approach has been taken when contemplating how pain relief will be applied in animal research protocols.

In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides.

Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models.

Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging.

In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.

From bench to cageside: Risk assessment for rodent pathogen contamination of cells and biologics.

Many newly developed animal models involve the transfer of cells, serum, or other tissue-derived products into live rodents. These biologics can serve as repositories for adventitious rodent pathogens that, when used in animal studies, can alter research outcomes and result in endemic outbreaks. This review includes a description of some of the biologics that have inadvertently introduced infectious agents into in vivo studies and/or resulted in endemic outbreaks. I also discuss the points of potential exposure of specific biologics to adventitious rodent pathogens as well as the importance of acquiring a complete developmental and testing history of each biologic introduced into a barrier facility. There are descriptions of specific cases of mycoplasma and lactate dehydrogenase-elevating virus (LDHV), two of the most common organisms that contaminate cells and cell byproducts. The information in this article should help investigators and animal resource program personnel to perform an appropriate risk assessment of biologics before their use in in vivo studies that involve rodents.

Development of molecular and cellular biomarkers of pain.

Observation of physiologic and behavioral responses is the main method used to assess pain in people and animals. These approaches are often difficult to objectively measure in laboratory rodents and provide no insight into associated molecular and cellular changes in the organism. To identify CNS markers for pain, we analyzed the gene expression profiles of midbrain sections of mice that had experienced either adjuvant injections in the footpad or partial sciatic nerve ligation (PSL), which are recognized models of inflammatory and neuropathic pain, respectively. The potential for pain-associated factors to be present in the blood and to affect other tissues was analyzed by monitoring the growth of various cell lines that were exposed to serum from these mice and to plasma from rats experiencing surgical pain and their respective controls. Adjuvant injection increased the transcription of 12 genes and decreased that of 38 genes by at least 2-fold, whereas PSL increased the transcription of 2 genes and decreased that of 23, with no overlap. Serum from mice with PSL stimulated the growth of the rat mammary tumor cell line RMT50. Similarly, plasma collected from rats after a painful surgical procedure promoted the growth of RMT50 and MDA-MB-235 cells. These results demonstrate that the gene expression profiles of brain tissue from mice exposed to painful stimuli vary depending on the nature of the stimulus, and that the growth of some mammary tumor cell lines can be affected by blood collected from rodents exposed to these stimuli.

Characterization of the effects of Bcl-2 and Bcl-xl deletion mutant expression in cell lines used for antibody production.

Strategies to maximize monoclonal antibody (MAb) yields by in vitro production methods entail that hybridoma cells be maintained at high density. Approaches to increase culture density and antibody yields from hybridomas by inhibiting apoptosis through over-expression of exogenous Bcl-2 family genes have produced variable results. In order to determine if expression of mutant forms of Bcl-2 and Bcl-xl could increase viable culture densities and batch MAb yields when compared to parental cell lines, recombinant delta loop deletion mutant of these apoptotic inhibitory genes were expressed in a myeloma and two hybridoma cell lines. Expression of either Bcl-2-delta or Bcl-xl-delta in P3x63Ag8.653 myeloma cells did not significantly increase viable cell densities in cultures over time. However, the rapid post-peak decline in viable cell density was significantly reduced in Bcl-xl-delta-expressing hybridoma cell lines 552 and 7.16.4 and in Bcl-2-expressing hybridoma 7.16.4. Significant increases in MAb yield were only observed in cultures of Bcl-xl-delta-expressing hybridoma 7.16.4. Annexin staining in hybridoma 7.16.4 confirmed that apoptosis was the primary means of cell death in this cell line, and expression of Bcl-2-delta and Bcl-xl-delta inhibited programmed cell death. These results suggest that cell viability in cultures can be improved by transfection and selection of hybridomas that express delta loop deletion mutant forms of Bcl-2 family genes; however, improvements in MAb yields are dependent upon the genetic background of each manipulated cell line.

Improving animal research with an institutional electronic discussion group.

To remain at the forefront of scientific discovery, investigators continually are challenged to apply new approaches, instruments, and models to their work. Research institutions work to foster the exchange of ideas and resources, but this objective becomes more difficult to meet as the organization's size and complexity increase. To facilitate communication among researchers that use mice in their work and to provide increased opportunities for resource sharing, an electronic discussion group was formed at The Johns Hopkins University. The discussion group (jhu-mousers) is restricted to individuals within the institution's three campuses, and its 145 subscribers comprise faculty (including veterinarians), postdoctoral fellows, graduate students, and technicians. During its beginning 2 1/2-year period, jhu-mousers has received 207 postings that include seminar announcements; resource information; requests for mice, equipment, biological reagents, and technical assistance; and responses to these requests. The value of the electronic mailing list is evidenced by the fact that 70% of requests for resources or technical help have received at least one response, and this figure is likely to be underestimated because off-line responses are not included. Because the mailing list provides opportunities for tissue sharing and is conducive to refining experimental procedures used in mice, its application promotes the use of alternatives in animal research. To promote and assist the development of animal-user discussion groups at other institutions, the administration, applications, and benefits of an electronic mailing list for mouse users are discussed here.

Advances in monoclonal antibody technology: genetic engineering of mice, cells, and immunoglobulins.

The ability to produce antibodies that are directed against specific antigens has played a crucial role in advancing scientific discoveries. Recombinant technologies have extended the application of antibodies beyond the research laboratory and into the clinic for the treatment of cancer and other diseases. Creative approaches using these technologies have been used to reduce the antibody to its minimal functional size, and/or make them bifunctional (immunotoxins), bispecific, or less immunoreactive (humanized). Additionally, mice that are engineered to generate antibodies of human genomic origin have been used to produce therapeutic antibodies and are being further developed. As the research and clinical demands for antibodies continue to increase, the development of improved resources (cell lines and animals) to improve production efficiency, generate larger repertoires, and deliver greater yields of antibodies is being explored, and advances in this area are discussed further in this review.

Tamoxifen resistance and Her2/neu expression in an aged, irradiated rat breast carcinoma model.

Clear links have been established between occupational or therapeutic radiation exposure and breast cancer. Tamoxifen chemoprevention following radiation exposure may be able to reduce the risk of developing breast cancer later in life. In order to model carcinogenesis in this setting, an in vivo model of tamoxifen chemoprevention and tamoxifen failure in a radiation-induced rat mammary carcinoma model was characterized. Two hundred and twenty-seven 60-day-old female rats received whole body or sham exposure to ionizing radiation. Thirty days later long-term, continuous, tamoxifen chemoprevention was initiated in half the population and all animals were monitored over three and a half years for the development of mammary tumors. Mammary tumors were surgically removed and carcinomas were histologically identified and characterized. Results showed that tamoxifen chemoprevention decreased the incidence and prolonged the latency of radiation-induced mammary carcinomas. However, many individuals receiving tamoxifen chemoprevention developed their first carcinoma very late in life. These carcinomas shared morphological features distinct from the majority of carcinomas that developed in the absence of tamoxifen chemoprevention. Analyses of cell lines established from these carcinomas and immunohistochemistry of tumor sections revealed that the highest levels of Her2/neu expression were associated with in vivo tamoxifen exposure. Treatment of rat mammary carcinoma cells with an anti-rat Her2/neu monoclonal antibody (MAb 7.16.4) inhibited cell growth and this effect was more pronounced in the presence of tamoxifen. These studies suggest that carcinoma growth driven by the Her2/neu pathway may be associated with tamoxifen chemoprevention failure in the rat mammary carcinoma model. Additionally, strategies combining targeted Her2/neu antibodies, vaccines or drugs with estrogen pathway modification may be more effective in reducing breast cancer chemoprevention failures.

Comparison of In Vitro Monoclonal Antibody Production Methods with an In Vivo Ascites Production Technique.

Economic, technical, legislative, and ethical issues influence decisions concerning alternatives to the use of animals in biomedical research. Since the development of cell hybridoma technology, production of ascites in mice has been a popular technique for generating high concentrations of monoclonal antibodies. However, the availability of several in vitro methods that can be performed in most laboratories make these alternative methods of monoclonal antibody production an attractive option. To evaluate the practicality of the use of in vitro techniques, three tissue culture methods and a technique for production of ascites were compared on the basis of yield, material costs, and time requirements. Analysis of results revealed that the time and material costs to produce 100 mg of monoclonal antibody 7.16.4 by inducing ascites in irradiated mice was similar or slightly more than that for tissue culture in standard plastic flasks and hollow fiber cartridge bioreactors; however, tissue culture in gas permeable bags was slightly less expensive than these methods in terms of cost and time. When ascites production in irradiated mice was compared with tissue culture in plastic flasks or gas permeable bags for monoclonal antibody 225, the in vitro methods were approximately five times more expensive than the ascites production technique. Information reported here outlines factors that should be considered and evaluated when choosing a monoclonal antibody production method. Furthermore, these results documented that in vitro technology for the production of monoclonal antibodies can be adapted by most conventional laboratories to provide sufficient resources for the most commonly performed experimental protocols.