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Magnetic resonance imaging (MRI) is a clinically relevant, real-time imaging modality that is frequently utilized to assess stroke type and severity. However, specific MRI biomarkers that can be used to predict long-term functional recovery are still a critical need. Consequently, the present study sought to examine the prognostic value of commonly utilized MRI parameters to predict functional outcomes in a porcine model of ischemic stroke. Stroke was induced via permanent middle cerebral artery occlusion. At 24 hours post-stroke, MRI analysis revealed focal ischemic lesions, decreased diffusivity, hemispheric swelling, and white matter degradation. Functional deficits including behavioral abnormalities in open field and novel object exploration as well as spatiotemporal gait impairments were observed at 4 weeks post-stroke. Gaussian graphical models identified specific MRI outputs and functional recovery variables, including white matter integrity and gait performance, that exhibited strong conditional dependencies. Canonical correlation analysis revealed a prognostic relationship between lesion volume and white matter integrity and novel object exploration and gait performance. Consequently, these analyses may also have the potential of predicting patient recovery at chronic time points as pigs and humans share many anatomical similarities (e.g., white matter composition) that have proven to be critical in ischemic stroke pathophysiology. The study was approved by the University of Georgia (UGA) Institutional Animal Care and Use Committee (IACUC; Protocol Number: A2014-07-021-Y3-A11 and 2018-01-029-Y1-A5) on November 22, 2017.

Intraspinal inflammatory and immune responses are considered to play central roles in the pathological development of spinal cord injury. This study aimed to decipher the dynamics of systemic immune responses, initiated by spinal cord injury. The spinal cord in mice was completely transected at T8. Changes in the in vivo inflammatory response, between the acute and subacute stages, were observed. A rapid decrease in C-reactive protein levels, circulating leukocytes and lymphocytes, spleen-derived CD4+ interferon-γ+ T-helper cells, and inflammatory cytokines, and a marked increase in neutrophils, monocytes, and CD4+ CD25+ FOXP3+ regulatory T-cells were observed during the acute phase. These systemic immune alterations were gradually restored to basal levels during the sub-acute phase. During the acute phase of spinal cord injury, systemic immune cells and factors showed significant inhibition; however, this inhibition was transient, and the indicators of these serious disorders gradually returned to baseline levels during the subacute phase. All experiments were performed in accordance with the institutional animal care guidelines, approved by the Institutional Animal Care and Use Committee of Experimental Animal Center of Drum Tower Hospital, China (approval No. 2019AE01040) on June 25, 2019.

Apoptosis is an important programmed cell death process involved in ischemia/reperfusion injury. MicroRNAs are considered to play an important role in the molecular mechanism underlying the regulation of cerebral ischemia and reperfusion injury. However, whether miR-670 can regulate cell growth and death in cerebral ischemia/reperfusion and the underlying mechanism are poorly understood. In this study, we established mouse models of transient middle artery occlusion and Neuro 2a cell models of oxygen-glucose deprivation and reoxygenation to investigate the potential molecular mechanism by which miR-670 exhibits its effects during cerebral ischemia/reperfusion injury both in vitro and in vivo. Our results showed that after ischemia/reperfusion injury, miR-670 expression was obviously increased. After miR-670 expression was inhibited with an miR-670 antagomir, cerebral ischemia/reperfusion injury-induced neuronal death was obviously reduced. When miR-670 overexpression was induced by an miR-670 agomir, neuronal apoptosis was increased. In addition, we also found that miR-670 could promote Yap degradation via phosphorylation and worsen neuronal apoptosis and neurological deficits. Inhibition of miR-670 reduced neurological impairments after cerebral ischemia/reperfusion injury. These results suggest that microRNA-670 aggravates cerebral ischemia/reperfusion injury through the Yap pathway, which may be a potential target for treatment of cerebral ischemia/reperfusion injury. The present study was approved by the Institutional Animal Care and Use Committee of China Medical University on February 27, 2017 (IRB No. 2017PS035K).

Sunitinib has been used as the first-line therapy to suppress the metastatic clear cell Renal Cell Carcinoma (ccRCC) as it could function via suppressing the tumor growth and angiogenesis. Yet most ccRCC tumors may still regrow due to the development of sunitinibresistance, and detailed mechanisms remain unclear. Here, we found the estrogen receptor β (ERβ) in RCC cells might function via increasing the endothelial cell tube formation to alter the sensitivity to sunitinib treatment. Mechanism dissection revealed that ERβ could function via transcriptional regulation with direct binding to the promoter to increase the cytokine Angiopoietin-2 expression in the ccRCC cells. The up-regulated Angiopoietin-2 could then increase the Tie-2 phosphorylation in the endothelial Huvec cells to promote the angiogenesis tube formation that results in increasing the endothelial cell resistance to sunitinib treatment. Preclinical study using an in vivo mouse RCC model also confirm in vitro data. Targeting this newly identified ERβ/Angiopoietin2/Tie-2 signaling-increased angiogenesis tube formation with the FDAapproved anti-estrogen, ICI 182,780 (Faslodex) may help in the development of a novel combined therapy with sunitinib to better suppress the ccRCC progression. Funding Statement: This work was partially supported by the URMC Urology research fund and the George Whipple Professorship Endowment. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: The Institutional Animal Care and Use Committee (IACUC) review board at University of Rochester Medical Center approved the studies.

BackgroundTumor associated macrophages (TAMs) play a critical role in tumor immunosuppression and resistance to immune checkpoint blockade. Reprogramming ‘M2’ TAMs to a proinflammatory ‘M1’ phenotype by selectively silencing M2 phenotype-driving transcription factors, such as STAT6, is a promising strategy to relieve TAM-induced immunosuppression. We have developed exoASO-STAT6™, an investigational therapeutic candidate consisting of exosomes loaded with antisense oligonucleotides (ASOs) targeting STAT6. By leveraging the TAM tropism of exosomes, exoASO-STAT6™ is the first systemically administered exosome designed to selectively silence STAT6 in TAMs. Preclinical biodistribution studies demonstrated that the liver is the main organ targeted by exoASO after intravenous (IV) dosing.MethodsWe evaluated the translational potential of exoASO-STAT6 to treat hepatocellular carcinoma through pharmacokinetics (PK), pharmacodynamic (PD) and anti-tumoral efficacy studies in preclinical models, as well as computational analysis of human HCC datasets.ResultsPK/PD were evaluated in naïve mice dosed IV with a single dose of exoASO-STAT6. PK analysis showed that the STAT6-ASO is rapidly cleared from plasma. Retention of the ASO in liver was dose-dependent and observed for at least 21 days. exoASO-STAT6 induced significant Stat6 mRNA knockdown (KD) in liver tissue with maximum KD at day 7 (70% KD at the 30 ug dose). IV administration of exoASO-STAT6 in an orthotopic, CPI resistant HCC model attenuated tumor growth and induced complete remission of tumor lesions in 50% of mice, while combination therapy with anti-PD1 antibodies further enhanced anti-tumor activity (75% complete remissions). Gene expression and histological analysis of the liver showed effective remodeling of the tumor microenvironment including a significant increase in interferon and cytotoxic T-cell gene signatures and iNOS expression. PD studies were also performed in cynomolgus monkeys that demonstrated a dose-dependent and durable silencing of STAT6 mRNA (50% and 31% at 1- and 3- weeks post-dose, respectively). STAT6 knockdown correlated with a reduction in STAT6 target genes, IL4R and EGR2, confirming modulation of the STAT6 pathway. Finally, we identified a STAT6 macrophage transcriptional signature and show high expression in human HCC tumors, both in immune cell-rich and TAM-rich/CD8 T-cell low tumors that correlates with worse survival.ConclusionsIn summary, we demonstrate that exoASO-STAT6 has a durable PK/PD profile in the liver of several species and potent antitumoral efficacy in a preclinical model of HCC. Furthermore, we identify an inverse correlation between the STAT6 macrophage signature and survival in human HCC tumors. Altogether our data support the systemic administration of exoASO-STAT6 as a promising therapy for liver cancer.Ethics ApprovalFor MiceMice were maintained and treated at the animal care facility of Codiak Biosciences in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (CB2017-001).Animal housing and experimental procedures (mice) were conducted according to the French and European Regulations and the National Research Council Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee of Oncodesign (Oncomet) approved by French authorities (CNREEA agreement N° 91).For cynomolgus monkeys:All animals were maintained and treated at the animal care facility of Altasciences in compliance with the Animal Welfare Act and recommendations set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council 2011).

Background: Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves’ disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease. Methods: To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies. Findings: Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of DIO3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvβ3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury. Interpretation: The study provided a mechanistic insight into how D3-αvβ3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves’ disease, explained a plausible link between thyroid disorder and NS. Funding: Funding support from American Thyroid Association (ATA) to NJT (ATA-2018-050.R1). Declaration of Interest: Jochen Reiser has patents on novel strategies for kidney therapeutics and stands to gain royalties from their commercialization. He is the co-founder of Walden Biosciences (Cambridge, MA, USA), a biotechnology company in which he has financial interest, including stock. Other authors have nothing to disclose and there are no competing or conflicting interests. Ethical Approval: All animal experiments were carried out according to the NIH’s Guide for the Care and Use of Experimental Animals (National Academies Press, 2011), and approved by the Institutional Animal Care and Use Committee (IACUC) at Rush University (Chicago, Illinois, USA). Human biopsy kidney sections from healthy donors and patients with FSGS, DN and MCD were procured in accordance with guidelines on human research and with approval of the Institutional Review Board of Rush University Medical Center (Chicago, Illinois, USA).

The purpose of this work was to determine the safety of inhaled hydrogen gas in healthy animals. Female mice were exposed to medical air with or without hydrogen gas (concentration 2.4%) for 72 hours (n = 25 mice/group). Mice underwent a standardized and validated neurobehavioral examination, SmithKline Beecham, Harwell, Imperial College, Royal London Hospital, Phenotype Assessment (SHIRPA) protocol, prior to and following the exposure period. Blood was withdrawn for serologic evaluation and all major organ tissues were evaluated histologically. The average hydrogen concentration within the chamber was 2.27%. Following exposure, there was no significant change in body weight in either group. Similarly, there was no significant change in the total SHIRPA score, although hydrogen-treated mice exhibited significantly lower spontaneous locomotor activity (P < 0.0001) in a subset of the test; all other aspects of the mouse neurologic exam were normal in hydrogen-treated animals. Brain histopathology was also normal in all mice, as was the histology of all other major organs. There were no significant differences in complete blood count, serum chemistry, or arterial blood gases between control and hydrogen-treated mice (P > 0.05 for all). Hydrogen gas did not appear to cause significant adverse effects when administered to healthy mice for 72 hours, with the possible exception of decreased spontaneous locomotor activity. The study was approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital, USA (approved number 18-01-3536) on January 25, 2018.

Calorie restriction (CR) has been well proved to be a powerful tool to improve metabolic health associated with aging; and many types of CR have been proposed. Intermittent CR has become a trend in recent years due to its better compliance than continuous CR every day. However, there are few studies that directly compare the interventional efficacy of intermittent CR to continuous CR in metabolic disorders such as diabetes. In this study, we analyzed two regimes of intermittent CR with the calorie-matched continuous CR in two diabetic mouse models including db/db and streptozotocin-treated mice. Intermittent CR was carried out by a fasting-mimicking diet (FMD, with 30% calorie intake of the control per day) for 2 days or 5 days (i.e., 2-5 or 5-9 regimes followed by free eating for 5 or 9 days respectively). In the two diabetic mouse models, both intermittent CR and continuous CR significantly reduced fasting blood glucose level and improved insulin sensitivity. However, intermittent CR performed significantly better than continuous CR in improving glycemic control and insulin sensitivity in db/db mice. In addition, intermittent CR improved the glucose homeostasis of the db/db mice without causing loss of body weight. Analyses with the pancreatic islets reveal that intermittent CR profoundly elevated the number of insulin-positive cells in both diabetic mouse models. In conclusion, our study indicated that both intermittent CR and continuous CR can lower fasting blood glucose level in the diabetic mice, while intermittent CR is better than the latter in improving insulin sensitivity. Funding Statement: This study was supported by research grants from the National Natural Science Foundation of China (31630036 to Y.C.), the Ministry of Science and Technology of China (2016YFA0500103 to Y.C.), and the Chinese Academy of Sciences (XDA12010102, QYZDJ-SSW-SMC008, ZDRW-ZS-2016-8, and Y817X11141 to Y.C Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) with an approval number 2010-AN-8.

Anterior cruciate ligament (ACL) injuries are frequent, as >200,000 injuries occur in the United States alone each year. Owing to the risks for associated meniscus and cartilage damage, ACL injuries are a significant source of both orthopedic care and research. Given the extended recovery course after ACL injury, which often lasts 1-2 years, and is associated with limited participation in sports and activities of daily living for patients, there is a critical need for the evolution of new and improved methods for ACL repair. Subsequently, animal models of ACL reconstruction (ACLR) play a key role in the development and initial trialing of novel ACL interventions. This article provides a clear operative description and associated illustrations for a validated, institutional animal care and use committee, and veterinarian approved and facile model of ACLR to serve researchers investigating ACLR.

Background: The drug-resistance and relapse of diffuse large B-cell lymphoma (DLBCL), which are related to mesenchymal stem cells (MSCs), have become increasingly common. However, the underlying mechanisms remain elusive. Methods: CCK 8 assay, colony formation assay, and xenograft mouse model were used to investigate the effects of hBMSCs on DLBCL growth.Immunohistochemistry, qRT-PCR, and ELISA were used to study the expressions of IL-6 and IL-17A. Flow cytometry was used to analyze Th17 cells and Treg cells expressions. Western blot analysis, microarray analysis, and bioinformatics analysis were used to analyze the pathways of IL-6 or IL-17A mediated DLBCL growth. Findings: HBMSCs promoted DLBCL growth by secreting IL-6 in vitro and in vivo and simultaneously upregulating IL-17A in vitro. IL-6 and IL-17A synergistically promoted the growth and drug-resistance of DLBCL cells by protecting them from spontaneous or drug-induced apoptosis in vitro. IL-6 or IL-17A activated the JAK2/STAT3 pathway or upregulated cyclin D2 via activation of PI3K/Akt signaling in vitro, respectively. Interpretation: The present results indicated that hBMSCs might have a "dual effect" on promoting DLBCL progression and drug-resistance by secreting IL-6 and upregulating IL-17A. IL-6, IL-17A, p-STAT3, p-Akt or cyclin D2 may be potential molecular targets for overcoming drug-resistance in patients with relapsed or refractory DLBCL. Funding Statement: This work was supported by the Natural Science Foundation of Guangdong Province, China (2018A0303130167); the Guangzhou Planned Project of Science and Technology, China (201707010279, 201300000100, and 201704020105); the Guangdong Medical Science and Research Foundation, China (A2018258); the Guangzhou General Science and Technology Project of Health and Family Planning, China (20181A011011). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This research was approved by the Ethics Committee of Guangzhou First People’s Hospital (K-2017-066-02). Written informed consent was obtained from all participants or their families. All animal experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee at Guangzhou First People's Hospital, Guangzhou Medical University.

While animal models of controlled cortical impact often display short-term motor dysfunction after injury, histological examinations do not show severe cortical damage. Thus, this model requires further improvement. Mice were subjected to injury at three severities using a Pin-Point™-controlled cortical impact device to establish secondary brain injury mouse models. Twenty-four hours after injury, hematoxylin-eosin staining, Fluoro-Jade B histofluorescence, and immunohistochemistry were performed for brain slices. Compared to the uninjured side, we observed differences of histopathological findings, neuronal degeneration, and glial cell number in the CA2 and CA3 regions of the hippocampus on the injured side. The Morris water maze task and beam-walking test verified long-term (14–28 days) spatial learning/memory and motor balance. To conclude, the histopathological responses were positively correlated with the degree of damage, as were the long-term behavioral manifestations after controlled cortical impact. All animal procedures were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University School of Medicine.

COVID-19 has proven to be a metabolic disease resulting in adverse outcomes in individuals with diabetes or obesity. Patients infected with SARS-CoV-2 and hyperglycemia suffer from longer hospital stays, higher risk of developing acute respiratory distress syndrome (ARDS), and increased mortality compared to those who do not develop hyperglycemia. We find the prevalence of hyperglycemia in patients with COVID-19 ARDS to be equal to those with non-COVID-19 ARDS. Nevertheless, the pathophysiological mechanism(s) of hyperglycemia in COVID-19 remains poorly characterized. Here we show that insulin resistance rather than pancreatic beta cell failure is the prevalent cause of hyperglycemia in COVID-19 patients with ARDS, independent of glucocorticoid treatment. A screen of protein hormones that regulate glucose homeostasis reveals that the insulin sensitizing adipokine adiponectin is reduced in hyperglycemic COVID-19 patients. Hamsters infected with SARS-CoV-2 also have diminished expression of adiponectin. Together these data suggest that adipose tissue dysfunction may be a driver of insulin resistance and adverse outcomes in acute COVID-19. Funding Information: This work was supported by the following grants: NIH R01 DK121140 (J.C.L.) and R01 DK121844 (J.C.L.), NCI R01 CA234614 (R.E.S), NIAID 2R01 AI107301 (R.E.S), and NIDDK R01 DK121072 (R.E.S). R.E.S. and J.C.L are supported as Irma Hirschl Trust Research Award Scholars. In addition, this study received support from NewYork-Presbyterian Hospital (NYPH) and Weill Cornell Medical College (WCMC), including the Clinical and Translational Science Center (CTSC) (UL1 TR 0002384). Declaration of Interests: R.E.S. is on the scientific advisory board of Miromatrix Inc. R.E.S. is a speaker and consultant for Alnylam Inc. The other authors have no conflict of interest. Ethics Approval Statement: The registry was approved by the institutional review board of WCMC (1405015116, 20-05022072, 20-03021681). All animal experiment procedures, breeding, and ethical use were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee at Mount Sinai School of Medicine.

With the recent upswing of infectious disease outbreaks (coronavirus, influenza, Ebola, etc), there is an ever-increasing need for biocontainment animal use protocols to better address the research of emerging diseases and to increase the health of both animals and humans. It is imperative that we as a research community ensure these protocols are conducted with the utmost scrutiny and regulatory compliance for the welfare of the animals as well as the health and safety concerns of the individual conducting these studies. Both the welfare of the animals and the health and safety of the research staff must be balanced with the integrity of the science being studied. Even prior to reviewing biocontainment protocols, the research stakeholders should have professional and collegial interactions across all levels of the proposed project. These stakeholders should include the attending veterinarian, the principal investigator, the sponsor, and any organic institutional health and safety assets (environmental health and safety, occupational health, biosafety personnel, medical personnel, facilities operations and maintenance, etc). At most institutions, these stakeholders are members of the Institutional Animal Care and Use Committee and may not possess the necessary tools to properly assess an Animal Biosafety Level 3 and 4 animal use protocol. It is the goal of this article to review some basic concepts of biocontainment, discuss critical communications and preapprovals, clinical observations, medical interventions and supportive care, scientific and study endpoints, euthanasia criteria, animal manipulations, documentation, training, emergency response and contingency plans, security, and decontamination and provide a scenario-based and informative thought-provoking process Institutional Animal Care and Use Committee members and veterinary staff may consider during Animal Biosafety Level 3 and 4 protocol review. These topics will enhance the ability of all stakeholders to balance the protection of the people with the integrity of the science and ultimately the welfare of the animal.

Recently approved vaccines have shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, and how boosting alters immunity to wildtype and newly emerging strains, remains incompletely understood. Here we profiled the humoral immune response in a cohort of non-human primates immunized with a recombinant SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a one or two-dose regimen with a saponin-based adjuvant Matrix-M™. While antigen dose had minimal effects, boosting significantly altered the humoral response, driving unique vaccine-induced antibody fingerprints. Differences in antibody effector functions and neutralization were associated with protection in the upper and lower respiratory tract, pointing to compartment-specific determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies targeting emerging SARS-CoV-2 variants. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants. Funding: This work was funded by Operation Warp Speed. We thank Colin Mann and Kathryn Hastie for production of Spike antigens. We thank Nancy Zimmerman, Mark and Lisa Schwartz, an anonymous donor (financial support), Terry and Susan Ragon, and the SAMANA Kay MGH Research Scholars award for their support. We acknowledge support from the Ragon Institute of MGH, MIT and Harvard, the Massachusetts Consortium on Pathogen Readiness (Mass CPR), the NIH (3R37AI080289-11S1, R01AI146785, U19AI42790-01, U19AI135995-02, U19AI42790-01, 1U01CA260476 – 01, CIVIC75N93019C00052), National Science Foundation Graduate Research Fellowship Grant No. #1745302, the Gates foundation Global Health Vaccine Accelerator Platform funding (OPP1146996 and INV-001650), and the Musk Foundation. Conflict of Interest: NP, MGX, JHT, BZ, SM, AMG, MJM, ADP, GG, GS, and LE are current or past employees of Novavax, Inc. and have stock options in the company. GA is the founder of Serom Yx Systems, Inc. AZ is a current employee of Moderna, Inc. but conducted this work before employment.Any opinion, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. YG, RC, MJG, CA, KMP, CL, DY, KB, MEM, JL, DM, CM, SS, FA, FK, EOS, DL, and MBF declare no competing interest. Ethical Approval: The work was conducted in accordance with a protocol approved by Texas Biomed’s Institutional Animal Care and Use Committee. All subjects signed informed consent and safety oversight was monitored by a data monitoring board.

Background Cancer immunotherapies are dependent on endogenous biomarker expression and other biological factors that often result in varying response rates across tumor types and benefit only a subset of patients. Conversely, conventional cytotoxics, the first-line treatment against solid tumors, are effective in a large patient population, but lack specificity, and often result in dose-limiting systemic toxicity. Here, we present SQ3370, a modular approach that activates doxorubicin (Dox) directly at the tumor site with reduced toxicity and potentially activates an immune response against tumor. The technology is independent of biomarkers, enzymatic activity, pH or oxygen levels and is hence expected to be effective in a wider group of patients. SQ3370 consists of a local intratumoral injection of a prodrug-capturing biomaterial (SQL70) followed by 5 daily infusions of SQP33, an attenuated prodrug of Dox. Complementary click chemistry groups in both components allow active Dox release at the tumor site (figure 1). SQP33 prodrug is ~82-fold less cytotoxic than Dox in vitro. We safely administered SQ3370 in dogs at 8.95-times the veterinary clinical dose of Dox, thus widening the therapeutic window, and showed minimal side effects including cardiotoxicity and immunosuppression. We hypothesize that releasing Dox at a local site with SQ3370 may also promote immune activation against the tumor. We evaluated this in a dual-tumor model of syngeneic MC38 tumors. Methods Immunocompetent mice were inoculated with MC38 tumor cells in two subcutaneous flanks. One tumor was intratumorally injected with the biomaterial, SQL70. SQP33 prodrug, control Dox, or saline was administered intravenously as per treatment schedule (figure 2A). Tumors harvested from a subset of mice at 2 weeks were assessed for immune biomarkers. Results In mice bearing two tumors, SQ3370 significantly increased overall survival and the anti-tumor response against injected tumors (figure 2B,C). Surprisingly, SQ3370 also induced regression of the non-injected tumors (figure 2D). Assessment of tumor-Infiltrating immune cells showed an increase in CD3+, CD4+, and CD8+ T cells and a decrease in regulatory T cells in both injected and non-injected lesions (figure 3). The T-cell response correlated with the anti-tumor efficacy data, supporting the immune activation hypothesis. Conclusions SQ3370 is a proof of concept example for a novel modular approach that addresses limitations of current immuno- and cytotoxic therapies for patients with solid tumors. Local release of Dox with SQ3370 expands the therapeutic window of Dox, minimizes toxicities and leads to a robust anti-tumor response that potentially also causes immune activation against the tumors. Acknowledgements The authors would like to thank the National Institutes of Health (NIH), the National Science Foundation (NSF), and Y Combinator. Ethics Approval This study, project number: SQI-FFS-ON-20181119_04_v4, was approved by the Institutional Animal Care and Use Committee (IACUC) of the vendor, following the guidance of Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), accreditation number 001516.

Background Although CAR T cells have been shown to be effective and potent in treating several hematologic malignancies, engineered T-cell therapies have had limited success in addressing solid tumors. Unlike liquid tumors where uniformly expressed antigens are accessible and can be effectively targeted, tumor access and antigen heterogeneity are a significant barrier to the successful development of CAR-T cells in solid tumors. Methods Here we demonstrate that the combination of a bi-specific T-cell engager (BiTE) targeting EpCAM with a CAR T cell targeting HER2 enhances the in vitro and in vivo anti-tumor activity against heterogenous solid tumors. Results We observed a dose-dependent enhancement of cytolytic activity when EpCAM-specific BiTEs were titrated alongside 4D5-based HER2-specific CAR T cells against HER2low tumors, enhancing maximal cytolysis by two-fold compared to CAR T cells alone (figure 1). Moreover, the escape of HER2low tumor cells in mixed heterogenous culture systems was circumvented by the combination of HER2-specific CAR T cells and EpCAM-specific BiTEs. The enhancement of efficacy was further demonstrated in an established HER2low MDA-MB-231 xenografts. HER2-specific CAR T cells were unable to contain Her2low tumors, whereas tumor growth was effectively controlled in mice receiving both EpCAM-specific BiTEs and HER2-specific CAR T cells. Conclusions Collectively, these data demonstrate that multi-antigen targeting mediated by BiTEs and CARs extends overall anti-tumor efficacy in preclinical models of heterogenous solid tumors. Fate Therapeutics is currently using its proprietary induced pluripotent stem cell (iPSC) product platform to generate iPSC-derived CAR T cells and iPSC-derived CAR NK cells that secrete BiTEs for the treatment of solid tumors. Ethics Approval These studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Background VISTA is a negative checkpoint regulator prominently expressed in the TME of a wide variety of cancers. In a preclinical model of colorectal cancer, monotherapy of small tumors (40 mm3) with anti-VISTA results in markedly slowed tumor growth. Mice bearing significantly larger tumors (600 mm3) are resistant to anti-PD-1 and anti-CTLA4 treatment and all mice die following treatment, indicating checkpoint resistance. Inclusion of anti-VISTA leads to complete rejection of 50% of tumors. Methods The underlying therapeutic mechanisms of leading to enhanced anti-tumor immunity in both models was investigated by high-dimensional scRNAseq of the CD45+ immune infiltrate of tumors 10 days after treatment initiation. Results In both modes, anti-VISTA treatment stimulated several pathways involving myeloid activation and antigen-presentation. Multi-spectral imaging of anti-VISTA treated tumors supported increased antigen presentation, and suppression assays showed that the myeloid infiltrate was less suppressive to T cells. Transcriptional analysis of tumor-specific CD8 T cells showed that anti-VISTA therapy induced T cell pathways highly distinct from the anti-exhaustion effects of anti-PD-1 therapy. Conclusions These data document the unique and complementary impact of targeting VISTA in contrast to PD-1 and CTLA-4 in both the myeloid and T cell lineages. These mechanistic insights strongly support the use of anti-VISTA to overcome the checkpoint resistance seen in contemporary treatments involving PD-1. Ethics Approval All mouse studies described in this work were carried out in accordance with the principles of the Guide for the Care and Use of Animals and were approved by the Institutional Animal Care and Use Committee of Dartmouth College, NH, USA (protocol 2012).

Background: Pertuzumab is currently used in combination with trastuzumab as the first-line treatment for HER2-positive metastatic breast cancer. However, pertuzumab was originally developed independently from trastuzumab and was later incidentally found to have synergistic efficacy when combined with trastuzumab, it remains to be seen whether a more potent synergistic efficacy partner exists for trastuzumab. Methods: A trastuzumab-based functional assay was used to screen anti-HER2 antibodies harboring trastuzumab-synergistic antitumor activity. The lead candidate 5G9, in combination with trastuzumab, was further characterized for its bioactivities in cell proliferation, cell apoptosis, antigen-antibody endocytosis and HER2-mediated cell signaling pathway blocking. Finally, animal models were used to evaluate the in vivo synergistic antitumor efficacy of 5G9 in combination with trastuzumab. Findings: Compared to pertuzumab, 5G9 demonstrated more potent synergistic cell growth inhibitory activity when combined with trastuzumab (85% vs. 55%, P< 0.001). In addition, 5G9 exhibited a higher internalization rate than pertuzumab (20% vs. 9%, P< 0.05), and was able to further synergize with trastuzumab to promote antigen-antibody endocytosis. The internalination rate of the combination of 5G9 and trastuzumab was higher than the combination of pertuzumab and trastuzumab (35% vs. 14%, P< 0.001). In vivo animal studies demonstrated that 5G9 in combination with trastuzumab showed more potent synergistic antitumor efficacy than the combination of pertuzumab and trastuzumab. Interpretation: 5G9, together with trastuzumab, may provide a potential opportunity for more efficacious treatment of HER2-positive cancers. Funding Statement: Chunni Zhang was supported by grants from National Natural Science Foundation of China (no. 81472021 and no. 81672102) and from the State Key Laboratory of Analytical Chemistry for Life Science (no. 5431ZZXM1907). The research was funded in part by Biosion Incorporated. Declaration of Interests: Xiaoyao Hao, Shukai Xia and Jinyu Liu and Mingjiu Chen are employees of Biosion Inc.. The remaining authors declare no competing interests. Ethics Approval Statement: All animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines and approved by the Animal Ethical Committee.

Background: Identifying effective drugs to suppress vascular inflammation is a promising strategy to delay the progression of abdominal aortic aneurysm (AAA). Itaconate has a vital role in regulating inflammatory activation in various inflammatory diseases. However, the role of itaconate in the progression of AAA is unknown. In this study, we explored the inhibitory effect of itaconate on AAA formation and its underlying mechanisms. Methods: Quantitative PCR, western blotting and immunohistochemistry were used to determine Irg1 and downstream Nrf2 expression in human and mouse AAA samples. Liquid chromatograph-mass spectrometry (LC-MS) analysis was performed to measure the abundance of itaconate. OI treatment and Irg1 knockdown were performed to study the role of OI in AAA formation. Nrf2 intervention in vivo were performed to detect the critical role of Nrf2 in the beneficial effect of OI on AAA. Findings: We found that itaconate suppressed the formation of angiotensin II (Ang II)-induced AAA in apolipoprotein E-deficient (Apoe-/-) mice, while Irg1 deficiency exerted the opposite effects. Mechanistically, itaconate inhibited vascular inflammation by enabling Nrf2 to function as a transcriptional repressor of downstream inflammatory genes via alkylation of Keap1. Moreover, Nrf2 deficiency significantly aggravated inflammatory factor expression and promoted AAA formation. In addition, Keap1 overexpression significantly promoted Ang II-induced AAA formation, which were inhibited by itaconate. Interpretation: Itaconate inhibited AAA formation by suppressing vascular inflammation, and therapeutic approaches to increase itaconate are potentially beneficial for preventing AAA formation. Funding: National Natural Science Foundations of China and Guangzhou regenerative medicine and Health Laboratory of Guangdong. Declaration of Interest: None. Ethical Approval: Human AAA samples were obtained from patients undergoing open surgical repair according to protocols approved by the Research Ethics Committees of Nanfang Hospital (ethical approval number: NFEC-2019-086). All animal protocols were approved by the Institutional Animal Care and Use Committee at Southern Medical University.

CRISPR (clustered regularly interspaced short palindromic repeats)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of sensitive intracellular sensors. Likewise, lymphocytes of the adaptive immune system are largely refractory to CRISPR-enhanced homology-directed repair (HDR) due to inefficient or toxic delivery of donor templates via transient transfection methods. To overcome these challenges and limitations, we developed three improved methods for CRISPR-based genome editing using a hit-and-run transient expression strategy to minimize off-target effects and generate more precise genome editing. Overall, our enhanced CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering are expected to be widely applicable not only in hematopoietic cells but also other mammalian cell types of interest.All animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional Animal Care and Use Committee.

In this article we introduce the concept of biological rhythms in fish welfare and critically discuss a neglected aspect: time-dependent issues. First we should consider that fish, like most living organisms, posses a clock that drives rhythmic biological functions. Therefore, fish stress responses are strongly affected by the time of stressor exposure (e.g. day/night) according to their daily pattern of behavior (diurnal/nocturnal). Furthermore, drugs affect fish differently depending of time of administration. For instance, commonly used substances such as anesthetics may have strikingly different effects depending on the time of day. MS-222 -the most widely used fish anesthetics- has fastest effects and higher toxicity when applied during daytime, but it is much less toxic at night. Surprisingly, no attention has been paid to this time-dependent factor when establishing dosages and protocols for fish anesthesia and euthanasia. In summary, there is a need to take into account properly the role of biological rhythms when discussing fish welfare issues and regulations by the Institutional Animal Care and Use Committee (IACUC). En este artículo se introduce el concepto de ritmos biológicos en el bienestar de peces, y se discute críticamente un tema olvidado: aspectos dependientes del tiempo. Primero debemos considerar que los peces, al igual que la mayoría de organismos vivos, poseen un reloj interno que dirige los ritmos biológicos. Por tanto, las respuestas al estrés de los peces están fuertemente afectadas por la hora de exposición al agente estresante (p.e. día/noche) en función de su patrón diario de comportamiento (diurno/nocturno). Además, los fármacos pueden afectar de forma diferente a los peces dependiendo de la hora de la administración. Por ejemplo, sustancias comúnmente usadas como los anestésicos pueden tener efectos llamativamente distintos dependiendo de la hora del día. El MS-222, el anestésico más ampliamente utilizado en peces, tiene efectos más rápidos y mayor toxicidad cuando es aplicado por el día, pero es mucho menos tóxico por la noche. Sorprendentemente, no se ha prestado atención a este factor temporal cuando se establecen dosis y protocolos para la anestesia o eutanasia de peces. En resumen, es necesario tener en cuenta adecuadamente el papel de los ritmos biológicos cuando se discute el bienestar de peces y su regulación por los comités de uso y cuidado de animales.

Background Impaired social interaction is one of the essential features of autism spectrum disorder (ASD). Our previous copy number variation (CNV) study discovered a novel deleted region associated with ASD. One of the genes included in the deleted region is ARHGEF10. A missense mutation of ARHGEF10 has been reported to be one of the contributing factors in several diseases of the central nervous system. However, the relationship between the loss of ARHGEF10 and the clinical symptoms of ASD is unclear. Methods We generated Arhgef10 knockout mice as a model of ASD and characterized the social behavior and the biochemical changes in the brains of the knockout mice. Results Compared with their wild-type littermates, the Arhgef10-depleted mice showed social interaction impairment, hyperactivity, and decreased depression-like and anxiety-like behavior. Behavioral measures of learning in the Morris water maze were not affected by Arhgef10 deficiency. Moreover, neurotransmitters including serotonin, norepinephrine, and dopamine were significantly increased in different brain regions of the Arhgef10 knockout mice. In addition, monoamine oxidase A (MAO-A) decreased in several brain regions. Conclusions These results suggest that ARHGEF10 is a candidate risk gene for ASD and that the Arhgef10 knockout model could be a tool for studying the mechanisms of neurotransmission in ASD. Trial registration Animal studies were approved by the Institutional Animal Care and Use Committee of National Taiwan University (IACUC 20150023). Registered 1 August 2015. Electronic supplementary material The online version of this article (10.1186/s13229-018-0197-5) contains supplementary material, which is available to authorized users.

Objectives: The purpose of the study is to assess both the approximate lethal dose and the single dose intramuscular injection toxicity of Danggui (Angelica gigantis radix) pharmacopuncture (DGP) in Sprague-Dawley (SD) rats. Methods: The experiments were conducted at the good laboratory practice (GLP) laboratory, Biotoxtech Co., which is a laboratory approved by the ministry of food and drug safety (MFDS). The study was performed according to the GLP regulation and the toxicity test guidelines of the MFDS (2009) after approval of the institutional animal care and use committee of Biotoxtech. Single doses of DGP were injected intramuscularly into the rats in three test groups of 6 week old SD rats (5 male and 5 female rats per groups) in the amounts of 0.1, 0.5, and 1.0 mL/animal for groups 2, 3, and 4, respectively, and normal saline solution in the amount of 1.0 mL/animal was injected intramuscularly into the rats (5 male and 5 female rats) in the control group. Observations of the general symptoms and weight measurements were performed during the 14 day observation period after the injection. Hematologic and serum biochemical examination, necropsy, and a local tolerance test at the injection site were done after the observation period. Results: No death was observed in three test groups (0.1, 0.5 and 1.0 mL/animal group). In addition, the injection of DGP had no effect on general symptoms, weights, hematologic and serum biochemical examination, and necropsy. The results from the local toler ance tests at injection site showed no treatment related effects in the SD rats. Conclusion: The results of single dose intramuscular injection of DGP suggest that the approximate lethal dose is above 1.0 mL/animal for both male and female SD rats and that intramuscular injection of DGP may be safe.

Purpose: To evaluate the usefulness of dynamic contrast-enhanced ultrasonography (DCE-US) in the early quantification of hemodynamic change following administration of the vascular disrupting agent (VDA) CKD-516 using a rabbit VX2 liver tumor model. Methods: This study was approved by our institutional animal care and use committee. Eight VX2 liver-tumor-bearing rabbits were treated with intravenous CKD-516, and all underwent DCE-US using SonoVue before and again 2, 4, 6, and 24 hours following their treatment. The tumor perfusion parameters were obtained from the time-intensity curve of the DCE-US data. Repeated measures analysis of variance was performed to assess any significant change in tumor perfusion over time. Relative changes in the DCE-US parameters between the baseline and follow-up assessments were correlated with the relative changes in tumor size over the course of seven days using Pearson correlation. Results: CKD-516 treatment resulted in significant changes in the DCE-US parameters, including the peak intensity, total area under the time-intensity curve (AUCtotal), and AUC during wash-out (AUCout) over time (Ptotal and AUC during wash-in (AUCin) seen on the two-hour follow-up were significantly lower than the baseline values (P Conclusion: Our results suggest that a novel VDA (CKD-516) can cause disruption of tumor perfusion as early as two hours after treatment and that the therapeutic effect of CKD-516 treatment can be effectively quantified using DCE-US.

PURPOSE: To prospectively evaluate quantitative diffusion magnetic resonance (MR) imaging for monitoring skeletal muscle injury and repair after femoral artery ligation in mice. MATERIALS AND METHODS: All experimental procedures were approved by the local institutional animal care and use committee. Muscle degeneration and regeneration were induced in 16 mice by using unilateral ligation of the femoral artery. Diffusion-tensor and T2-weighted MR imaging examinations were performed before, immediately after, and 3, 10, and 21 days after ligation. Histologic analysis was also performed at these time points. The dynamic changes in T2 and in five diffusion-tensor imaging indexes were studied by using histogram analysis. Differences between the ligated and nonligated limbs were assessed with paired t tests, and analysis of variance was used to determine temporal evolutions. Parametric maps were clustered to depict regional differences in the responses of the different MR imaging indexes. RESULTS: MR indexes in the ligated limb changed over time (P < .007), and temporal evolutions in the ligated and nonligated limbs differed significantly (P < .001). When ischemia was induced, diffusivity and T2 increased, with a maximum change at 3 days, when most muscle damage was observed at histologic analysis. At 10 days, diffusion values were reduced overall, whereas T2 was still increased. At 21 days, parameter values had largely returned to normal. Changes on the diffusion-tensor and T2 maps had spatial differences, which corresponded to the different phases of tissue regeneration observed at histologic analysis. An additional finding was the transient change in direction of the principal eigenvector during the period of maximal muscle damage. CONCLUSION: After femoral artery ligation, the diffusion-tensor indexes changed dynamically in association with the severity and location of muscle damage.

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in rodent models of brain injury is lacking. Borrowing from the operant literature our laboratory utilized a basic scent discrimination paradigm 1-4 in order to assess deficits in frontally-injured rats. Previously we have briefly described the Dig task and demonstrated that rats with frontal brain damage show severe deficits across multiple tests within the task 5 . Here we present a more detailed protocol for this task. Rats are placed into a chamber and allowed to discriminate between two scented sands, one of which contains a reinforcer. The trial ends after the rat either correctly discriminates (defined as digging in the correct scented sand), incorrectly discriminates, or 30 sec elapses. Rats that correctly discriminate are allowed to recover and consume the reinforcer. Rats that discriminate incorrectly are immediately removed from the chamber. This can continue through a variety of reversals and novel scents. The primary analysis is the accuracy for each scent pairing (cumulative proportion correct for each scent). The general findings from the Dig task suggest that it is a simple experimental preparation that can assess deficits in rats with bilateral frontal cortical damage compared to rats with unilateral parietal damage. The Dig task can also be easily incorporated into an existing cognitive test battery. The use of more tasks such as this one can lead to more accurate testing of frontal function following injury, which may lead to therapeutic options for treatment. All animal use was conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee.

Can it ever be ethical to leave pain untreated in laboratory animals? Despite advances in the development of alternatives to using animals in research, scientists still often cite a need to use live animals in experiments. Once that need has been confirmed by funding agencies and/or the local IACUC (Institutional Animal Care and Use Committee) or other ethics committee, but before the research plans are finalized, the potential for animal pain and distress must be assessed, and plans laid to minimize animal suffering. Regulatory concern for the pain of laboratory animals is not new. In 1970, the United States Congress updated the Animal Welfare Act (AWA) in calling for “adequate veterinary care, including the appropriate use of anesthetic, analgesic or tranquilizing drugs, when such use would be proper in the opinion of the attending veterinarian” [1]. This policy is echoed in a 2009 report of the National Academy of Sciences on the pain of laboratory animals, expanding pain management to a more generalized obligation than simply an aspect of the veterinarian's duties: “Laboratory animals need not experience substantial or ongoing pain and . . . prevention and alleviation of pain is an ethical imperative” [2]. The eighth edition of the Guide for the Care and Use of Laboratory Animals similarly states that “institutions are expected to provide oversight of all research animals and ensure that pain and distress are minimized” [3]. The ethical principle underlying laboratory animal welfare policy is that causing pain and distress to sentient animals is permissible, but requires strong justification [4]. It is a nuanced norm: causing pain is not categorically prohibited; it is allowed only with the justification that a valuable scientific experiment requires that animal pain be left untreated. Taking laboratory animal pain seriously does not equate to demonstrating a zero tolerance for animal pain. Without question, present public policy allows humans to cause laboratory animals unalleviated pain. The AWA, the Guide for the Care and Use of Laboratory Animals, and current Public Health Service policy all allow for the conduct of what are often called “Category E” studies – experiments in which animals are expected to undergo significant pain or distress that will be left untreated because treatments for pain would be expected to interfere with the experiment [3], [5], [6]. One example among many would be studies of new painkillers for arthritis, in which a control cohort of animals are left untreated, while the experimental groups receive the test painkillers (which themselves may prove not to give pain relief). But how are we to determine when to allow these experiments, or what limits to set on them? To move the mandate for “pain management when possible” from platitude to real-world guidance on difficult decisions requires carefully engaging the question of Category E experiments. In this paper we assume that animal research will continue into the foreseeable future and that if regulations remain largely as they are, there will be times when scientists, veterinarians, and the local IACUC will agree that some pain will be left untreated. We discuss how that commonly happens, how things have changed over the years since the publication of Russell and Burch's seminal work, and close with some suggestions on how the process might be improved.

International audience; PURPOSE: To prospectively evaluate in rats whether magnetic cell labeling can be used to noninvasively assess the technical success of endovascular cell therapy for abdominal aortic aneurysms (AAAs). MATERIALS AND METHODS: The study was approved by an institutional animal care and use committee. Vascular smooth muscle cells (VSMCs) labeled with iron oxide nanoparticles were seeded endovascularly in already formed AAAs. T2*-weighted gradient-echo and T2-weighted spin-echo magnetic resonance (MR) imaging was performed in vivo at 1.5 T before and 30 minutes after the injection of iron-loaded VSMCs (14 rats), nonlabeled VSMCs (three rats), or iron-free particles (three rats). Ten rats were euthanized shortly after the injection (day 0). Of the 10 remaining rats, which were seeded with iron-loaded cells, three were imaged on day 7 after cell delivery; three, on day 14; and four, on day 28; then they were euthanized. Ex vivo high-field-strength MR imaging of two AAAs was performed 28 days after cell delivery. Histologic examination of cross sections of all AAAs was performed. Statistical evaluations were performed with a nonparametric Wilcoxon correlation test. RESULTS: Magnetic cell labeling did not alter the capability of VSMCs to stabilize the diameter of the aneurysms. T2*-weighted gradient-echo images showed areas of hypointense signal within the aortic wall immediately and up to 1 month after cell therapy. The mean signal intensity decreased significantly after cell delivery (from 2362 +/- 244 [standard deviation] before to 434 +/- 275 after delivery, P < .001). Areas of hypointense signal and iron-loaded VSMCs were colocalized in the area of aortic wall reconstruction at both high-field-strength MR imaging and histologic analysis. CONCLUSION: MR imaging with magnetic cell labeling can be used to document endovascular cell delivery in already formed AAAs in rats.

Animal inoculation remains essential for many aspects of leptospirosis research. Although pathogenic leptospiral strains may be able to infect a wide variety of animals, the Golden Syrian hamster is the preferred model because its susceptibility to infection and the reproducibility of the results. Common applications include determination of strain infectivity, restoring virulence to culture-attenuated strains, assessing usefulness of potential vaccines or diagnostic antigens, and examining pathology of mammalian infection. In many respects, acute leptospirosis in the hamster reproduces the severe form of human leptospirosis (see Commentary). This unit provides a detailed description of the procedures involved in the hamster model of leptospirosis, including housing and handling of hamsters and intraperitoneal challenge (Basic Protocol 1), and monitoring response to challenge (Basic Protocols 2 and 3, and Alternate Protocol). Methods are provided for demonstrating infection, including culture isolation of leptospires from blood and tissues (Basic Protocol 2) and serologic studies and histopathology (Basic Protocol 3). Quantitative PCR is provided as an alternative detection method (Alternate Protocol). Although leptospires disseminate to many organs, the tissues of the liver and kidney are the primary targets of infection. The liver is heavily infected during the initial stage of hematogenous dissemination. Persistent infection occurs primarily in the kidneys of animals that survive acute disease. Infection of the renal tubules leads to shedding of infectious organisms in the urine, which is the primary mode of transmission in nature. A safety policy memorandum is provided (see Strategic Planning) that should be read and signed by all staff. In the Commentary section, a detailed rationale is presented for use of hamsters as an animal model of leptospirosis. The effects of challenge dose on the hamster model, LD50, in vitro passage of leptospiral strains, and immunological maturity are discussed. Alternative animal models are evaluated with reference to the concept of accidental versus reservoir hosts. The advantages and disadvantages of the hamster model are considered, with reference to use of alternative host animals to address specific research or vaccine-development issues. CAUTION: Pathogenic Leptospira species are Biosafety Level 2 (BSL-2) pathogens. L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii are known to be pathogenic for humans. Certain strains of L. inadai, L. meyeri, L. fainei, and L. alexanderi may also be pathogenic. L. biflexa, L. wolbachii, and L. parva are thought to be nonpathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. CAUTION: Protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals. This experiment requires Animal Biosafety Level 2 (ABSL-2) conditions. Follow all appropriate guidelines for the use and handling of infected animals. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. Fluids and tissues of infected animals are highly infectious. Numerous laboratory-associated infections, including deaths, have been reported (Miller et al., 1987; Pike, 1976). A safety policy memorandum is provided that should be signed by all staff (see Strategic Planning).