Charles River Laboratories Research Articles

Abstract P357: Renal Male and Female Cortical Mitochondria Display Bioenergetic Differences in Response to Varying Substrates

Introduction: In general, renal disease susceptibility is higher in males than in age-matched females until menopause. The damage to renal proximal tubules (PT) is key to the onset of kidney disease development due to their high capacity for reabsorption and intense mitochondrial metabolism. Our previous data identified higher oxygen consumption in the renal cortex of healthy male Sprague Dawley rats and lower H 2 O 2 production compared to females. We hypothesize that sex differences in renal mitochondrial substrate reliance trigger differential bioenergetic responses in PTs, which contributes to kidney disease development later in life. Methods: 11-week-old male and female Sprague Dawley rats were acquired from Charles River Labs. The kidneys were flushed, and the renal cortex was used for ex vivo experiments. Mitochondria were isolated with differential centrifugation, and rovided with different substrate combinations: 10mM pyruvate and 2mM malate (PM), 5mM of glutamate, malate, and succinate (GMS), or 5mM succinate only (S). Spectrofluorimetry was employed to measure membrane potential (TMRM) and H 2 O 2 production (Amplex Red), XF24e Seahorse was used for oxygen consumption rate (OCR). OriginPro was used for statistical analysis (one-way ANOVA with post hoc test) and data displayed as mean ± SEM. Results: Seahorse revealed that male renal cortical mitochondria exhibit higher basal and maximum OCR compared to females when provided with the GMS or PM substrate combinations (39.4 ± 1.18 vs 26.5± 0.88, respectively for basal GMS, and 52.0 ± 2.33 vs 32.6 ± 1.24 maximum respiration GMS, p<0.001). However, similar OCR was observed when mitochondria were provided succinate only (31.2 ± 1.67 vs 28.7 ± 1.30, respectively for basal and 50.5 ± 2.49 vs 51.1 ± 1.76 maximum respiration, p>0.05). Mitochondrial membrane potential was higher in females compared to males when succinate only was provided (p<0.001), whereas similar mitochondrial membrane potential was observed between males and females when GMS or PM were used. Regardless of the substrate combination used, H 2 O 2 production was higher in female renal mitochondria compared to males (p<0.001). Conclusions: We report that male and female renal cortical mitochondria display unique bioenergetic properties in response to varying substrates. These basal bioenergetic differences may contribute to the difference in susceptibility to kidney diseases later in life.

Mouse diet and vendor impact microbiome perturbation and recovery from early-life pulses of amoxicillin

The gut microbiome is a dynamic ecosystem shaped by various factors, including diet, sex, and environment. This system plays a crucial role in host health, such that perturbation in the form of antibiotics can lead to a vast array of negative outcomes. Accordingly, a growing body of work seeks to develop interventions to protect the microbiome during antibiotic exposure. While it is well established that antibiotics can disrupt the microbiome in the short term, how the impact of antibiotics is modulated by factors such as diet, sex, and environment is poorly understood. In this study, we analyzed how sex, diet and early life environment (vendor of origin) modulate the impact and recovery of the microbiome in mice treated with oral amoxicillin. Utilizing 16S rRNA gene sequencing and bioinformatic analyses, we looked at the microbiome response to antibiotics under high-sugar and high-fat (Western) and standard high-fiber mouse (Chow) diets in male and female C57BL/6 from Jackson Laboratory, and female mice from Charles River Laboratories. The microbiome composition of each set of mice had a distinct pre-antibiotic starting point, depending on vendor, sex, and diet. These differences were further exacerbated by antibiotic exposure and revealed that each group responded differently to this perturbation. In particular, we found that the Western diet microbiome had an exacerbated response to antibiotics with greater changes in alpha, and beta diversity, and microbial composition when compared to the antibiotic-treated Chow diet cohort. In particular, we detected blooms in Enterobacteriaceae, Streptococcaceae, and Peptostreptococcaceae that were not found in the Chow diet. The response to antibiotics on each diet also appeared to be vendor and sex dependent. Charles River female mice had less Bifidobacteriaceae, Clostridia_UCG.014, and Clostridiaceae compared to Jackson Laboratory females in a Western diet, while female mice had more Bacteroides, Bilophila, and Parasutterella compared to male mice. In a narrow sense, these findings underscore the importance of considering vendor source, diet, and sex when examining antibiotics’ impact on mice. The broader implications suggest that we will likely need to utilize patient-specific microbiome-informed approaches in the development of human therapeutics to safeguard the microbiome during antibiotic exposure.

Super-Resolution Ultrasound Imaging for Monitoring the Therapeutic Efficacy of a Vascular Disrupting Agent in an Animal Model of Breast Cancer.

Evaluate the use of super-resolution ultrasound (SRUS) imaging for the early detection of tumor response to treatment using a vascular-disrupting agent (VDA). A population of 28 female nude athymic mice (Charles River Laboratories) were implanted with human breast cancer cells (MDA-MB-231, ATCC) in the mammary fat pad and allowed to grow. Ultrasound imaging was performed using a Vevo 3100 scanner (FUJIFILM VisualSonics Inc) equipped with the MX250 linear array transducer immediately before and after receiving bolus injections of a microbubble (MB) contrast agent (Definity, Lantheus Medical Imaging) via the tail vein. Following baseline ultrasound imaging, VDA drug (combretastatin A4 phosphate, CA4P, Sigma Aldrich) or control saline was injected via the placed catheter. After 4 or 24 hours, repeat ultrasound imaging along the same tumor cross-section occurred. Direct intratumoral pressure measurements were obtained using a calibrated sensor. All raw ultrasound data were saved for offline processing and SRUS image reconstruction using custom MATLAB software (MathWorks Inc). From a region encompassing the tumor space and the entire postprocessed ultrasound image sequence, time MB count (TMC) curves were generated in addition to traditional SRUS maps reflecting MB enumeration at each pixel location. Peak enhancement (PE) and wash-in rate (WIR) were extracted from these TMC curves. At termination, intratumoral microvessel density (MVD) was quantified using tomato lectin labeling of patent blood vessels. SRUS images exhibited a clear difference between control and treated tumors. While there was no difference in any group parameters at baseline (0 hour, P > .09), both SRUS-derived PE and WIR measurements in tumors treated with VDA exhibited significant decreases by 4 (P = .03 and P = .05, respectively) and 24 hours (P = .02 and P = .01, respectively), but not in control group tumors (P > .22). Similarly, SRUS derived microvascular maps were not different at baseline (P = .81), but measures of vessel density were lower in treated tumors at both 4 and 24 hours (P < .04). An inverse relationship between intratumoral pressure and both PE and WIR parameters were found in control tumors (R2 > .09, P < .03). SRUS imaging is a new modality for assessing tumor response to treatment using a VDA.

Low to moderate doses of dietary resistant starch and a renin-angiotensin system inhibitor exhibit nephroprotective effects through similar mechanisms in a preclinical model of diabetes

The investigative goal of our study was to determine the molecular mechanisms of nephroprotection of a drug-diet intervention for type II diabetes in a pre-clinical model. Our working hypothesis proposed that a combination of moderate doses of dietary resistant starch and a low-dose renin-angiotensin system (RAS) inhibitor would decrease renal biomarkers associated with the progression and complications of type II diabetes through the modulation of the renal renin-angiotensin system. Four weeks old Zucker Diabetic Fatty rats ( fa/fa) ( ZDF, n=48) and Zucker lean rats (n=7) were obtained from Charles River Laboratories and acclimated for one week. ZDF rats were then randomly assigned to one of three diets: AIN-93G diet with 0% resistant starch (RS), which served as the control diet, AIN-93G diet with 5% RS, or AIN-93 with 10% resistant starch, which were adjusted at the expense of cornstarch in the diet composition, for a total of eight weeks. At six weeks old, they were randomly assigned to daily intraperitoneal (IP) injections of either captopril (at a dose of 0.5 g/kg of body weight) in saline water and ascorbic acid solution or placebo solution, which consisted of just the vehicle, until the end of the 8-weeks. Zucker lean rats were assigned to an AIN-93G diet with 0% RS and administered daily IP placebo injections at the same timeline as the ZDF counterparts. Rats were placed in metabolic cages 12 hours before euthanization by exsanguination to collect urine and feces. Samples and tissues were aliquoted and frozen, respectively, until further analyses. Enzyme-linked immunosorbent assays were used to quantify different renal biomarkers in the urine and serum samples (i.e., 25-hydroxy-vitamin D, albumin, creatinine). Protein was extracted from kidney samples for Western Blot analyses. ZDF 10% RS and captopril group (ZDF 10% cap) had a decrease of 62.5% in urinary protein creatinine ratio, 29% of urinary albumin levels, and 18% of 25-hydroxy vitamin D (25D) levels, compared to the diabetic control group (ZDF 0% RS placebo). The ZDF 10% RS cap group also exhibited a 62% increase in serum 25D levels, while the 5% RS and captopril group had an 8.7% increase in 1,25-dihydroxy-vitamin D levels compared to the diabetic control. The ZDF 10% RS cap group also presented a 5.5% increase in relative abundance of nephrin and a decrease of 5.9% and 33.3% in angiotensin II receptor I and aldosterone synthase relative abundance, respectively, compared to the diabetic control group. Our results suggest that a combination of dietary resistant starch and a RAS inhibitor help to protect kidney integrity in type II diabetic rats, evidenced by a decrease of biomarkers in the urine and expression of proteins that play a part in the RAS, and increase in serum biomarkers and expression of glomerular structural proteins. Our work was funded by a USDA-NIFA grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differences in Oxygen-Induced Retinopathy Susceptibility Between Two Sprague Dawley Rat Vendors: A Comparison of Retinal Transcriptomes

Purpose To determine the retinal transcriptomic differences underlying the oxygen-induced retinopathy phenotypes between Sprague Dawley rat pups from two commonly used commercial vendors. This will allow us to discover genes and pathways that may be related to differences in disease severity in similarly aged premature babies and suggest possible new treatment approaches. Methods We analyzed retinal vascular morphometry and transcriptomes from Sprague Dawley rat pups from Charles River Laboratories and Envigo (previously Harlan). Room air control and oxygen-induced retinopathy groups were compared. Oxygen-induced retinopathy was induced with the rat 50/10 model. Results Pups from Charles River Laboratories developed a more severe oxygen-induced retinopathy phenotype, with 3.6-fold larger percent avascular area at P15 and twofold larger % neovascular area at P20 than pups from Envigo. Changes in retinal transcriptomes of rat pups from both vendors were substantial at baseline and in response to oxygen-induced retinopathy. Baseline differences centered on activated pathways of neuronal development in Charles River Laboratories pups. In response to oxygen-induced retinopathy, during the neovascular phase, retinas from Charles River Laboratories pups exhibited activation of pathways regulating necrosis, neuroinflammation, and interferon signaling, supporting the observed increase of neovascularization. Conversely, retinas from Envigo pups showed decreased necrosis and increased focal adhesion kinase signaling, supporting more normal vascular development. Comparing oxygen-induced retinopathy transcriptomes at P15 to those at P20, canonical pathways such as phosphate and tensin homolog, interferon, and coordinated lysosomal expression and regulation element signaling were identified, highlighting potential novel mechanistic targets for future research. Conclusion Transcriptomic profiles differ substantially between rat pup retinas from Charles River Laboratories and Envigo at baseline and in response to oxygen-induced retinopathy, providing insight into vascular morphologic differences. Comparing transcriptomes identified new pathways for further research in oxygen-induced retinopathy pathogenesis and increased scientific rigor of this model.

90. The Diagnosis of Ventilator-associated pneumonia (DIVA) trial: Results of a Clinical Trial of a Bundled VAP Diagnostic Stewardship Intervention

Abstract Background Ventilator-associated pneumonia (VAP) is commonly overdiagnosed &amp; a primary driver of antibiotic overuse within intensive care units (ICUs). Antimicrobial stewardship programs have successfully leveraged diagnostic stewardship interventions (DSIs) to prevent overdiagnosis/overtreatment of diverse clinical syndromes, yet this approach has not been extended to VAP. In this trial, we aimed to evaluate the safety, feasibility &amp; efficacy of a novel DSI care bundle (DSI-CB) targeting VAP. Methods The DIVA trial (NCT05176353) was a pilot/feasibility trial conducted in 2 ICUs at Michigan Medicine from February 2022-February 2023. A DSI-CB targeting the respiratory culture testing pathway was implemented sequentially in study ICUs, using an interruptive electronic health record clinical decision support tool &amp; modifications to microbiology laboratory workflows (Table 1). Providers were counseled on DSI-CB use during bimonthly educational sessions during trial rollout &amp; via monthly email reminders thereafter. Rates of prespecified co-primary safety and secondary efficacy outcomes were compared between the postintervention study cohort &amp; 5-year preintervention historical controls using negative binomial regression. Interrupted time series analysis was used to evaluate ICU antibiotic utilization rates (ICU-AURs) to account for temporal trends. Results 1810 patients were admitted to study ICUs following DSI-CB implementation, 29% of whom were eligible for DSI-CB use. DSI-CB was utilized in 77% of eligible patients. Patient demographics, comorbidities &amp; measures of acute severity of illness were similar pre- &amp; post-intervention. DSI-CB implementation was not associated with increases in primary adverse safety outcomes (Table 2). DSI-CB implementation was associated with significant reductions in rates of total respiratory cultures ordered, rates of positive respiratory cultures &amp; reductions in both total &amp; broad-spectrum ICU-AURs (Table 2, Figure 1). Conclusion Implementation of a novel VAP DSI-CB was safe, feasible &amp; associated with significant reductions in rates of respiratory culture positivity and ICU-AURs. This represents the first trial of a DSI targeting VAP &amp; a novel avenue for ICU antimicrobial stewardship. Large-scale trials are warranted. Disclosures Owen Albin, MD, Biomeriux: Grant/Research Support|Charles River Laboratories: Advisor/Consultant|Shionogi: Advisor/Consultant Weirauch J. Andrew, RRT, Drager Medical: Equipment loaned for research purposes, but no monetary funds were provided.|Jones and Bartlett Learning: Honoraria Krishna Rao, MD, MS, Merck &amp; Co.: Grant/Research Support|Rebiotix: Advisor/Consultant|Seres Therapeutics: Advisor/Consultant|Summit Therapeutics: Advisor/Consultant Keith S. Kaye, MD, MPH, Entasis: Advisor/Consultant|Entasis: Honoraria|GSK: Advisor/Consultant|GSK: Honoraria|Merck: Advisor/Consultant|Merck: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Honoraria|VenatoRx: Advisor/Consultant|VenatoRx: Honoraria

2599. Impact of Alveolar Neutrophilia on Outcomes in Patients with Suspected Ventilated Hospital-Acquired Bacterial Pneumonia

Abstract Background Ventilated hospital-acquired bacterial pneumonia (vHABP) is a major source of morbidity &amp; mortality in hospitalized patients. Clinical risk scores and systemic biomarkers have limited clinical &amp; prognostic utility in vHABP. Alveolar biomarkers—specifically alveolar neutrophilia (AN), the percentage of polymorphonuclear cells (PMN%) present in bronchoalveolar lavage (BAL) fluid—gauge the host inflammatory response directly at the site of infection, offering an attractive but untested predictive risk biomarker in vHABP. We aimed to generate preliminary data exploring AN as a vHABP biomarker. Methods We performed a retrospective cohort study of intensive care unit (ICU) patients with vHABP from 2010-2023. vHABP was defined as development of acute hypoxic respiratory failure requiring invasive mechanical ventilation, clinical suspicion of pneumonia by treating teams prompting performance of BAL respiratory culture, and BAL PMN% &amp;gt; 50%. Patient demographic characteristics, comorbidities and acute severity of illness metrics were abstracted via chart review. PMN% values were dichotomized using median split rounded to the nearest PMN% decile ( &amp;gt; or &amp;lt; 80% PMN% in BAL fluid). Study outcomes included ventilator-free days, ICU length of stay and vHABP-directed antibiotic treatment duration within 30 days of vHABP. Results 44 patients met study inclusion criteria. 30 (68%) had a BAL neutrophil of &amp;gt; 80%. Patients with BAL PMN &amp;gt; 80% had a numerically higher use of tracheostomy and trend towards lower P/F ratio at the time of vHABP diagnosis [Table 1]. BAL PMN &amp;gt; 80% was associated with a 15% decrease in ventilator-free days (p &amp;lt; 0.01) and a 25% longer ICU length of stay (p &amp;lt; 0.01) than those with BAL PMN &amp;lt; 80% [Figure 1]. There was no significant difference in post-BAL vHABP-directed antibiotic duration in patients with and without BAL PMN &amp;gt; 80%. Conclusion Though limited by low sample size, this exploratory study suggests AN may be a potentially useful predictive risk biomarker in vHABP. Larger-scale, prospective studies of alveolar biomarkers—including AN—in vHABP are warranted. Disclosures Owen Albin, MD, Charles River Laboratories: Advisor/Consultant|Shionogi: Advisor/Consultant

SAT021 Development Of Niclosamide Analogs For Treatment Of Androgen Receptor Positive Hepatocellular Carcinoma

Abstract Disclosure: E.J. Montgomery: None. E. Xing: None. A. Dauki: None. H. Radomska: None. S.K. Kulp: None. L. Granchie: None. P. Li: None. C.C. Coss: None. Introduction: Hepatocellular carcinoma (HCC) is the predominant form of liver cancer and a leading cause of cancer death worldwide. HCC is also highly sexually dimorphic with males having a 2-4 times higher risk of HCC occurrence than females regardless of the disease etiology. Based upon the discrepancy in HCC incidence between the sexes, the role of sex hormone receptors, such as the androgen receptor (AR), in HCC progression has been investigated. Despite the evidence behind AR’s role in HCC lethality, clinical trials of anti-androgen therapy in HCC showed no significant clinical benefit. We recently reported the presence of abundant ligand-independent AR splice variants (AR-SVs) in HCC. Niclosamide is an anthelmintic drug that was identified in drugs screens for AR-V7 degrading and anti-HCC properties making it well-positioned as a potential therapeutic for AR/AR-SV positive HCC. Despite these features, niclosamide suffers from poor bioavailability by design resulting in dose limiting toxicities. Objective: 1) Identify and understand key functional groups of niclosamide for development of niclosamide analogs. 2) Develop niclosamide analogs towards improving bioavailability and limiting first pass metabolism. Methods: HCC cell lines were treated with niclosamide to determine impacts on AR expression, cell proliferation, and cell invasion. Mouse pharmacokinetics studies to evaluate drug parameters were conducted through Charles River Laboratories. Toxicity studies on a valine conjugate of niclosamide were carried out in mice over a two-week span using a range of drug concentrations. Results: Numerous niclosamide analogs were evaluated resulting in a valine conjugated niclosamide with improved bioavailability, a niclosamide analog with improved first pass metabolism, and its valine conjugate. Valine conjugated niclosamide showed improved oral exposure without resulting in toxicities. Conclusions: Niclosamide is a drug showing key activity against AR and HCC that lends itself to treatment of AR/AR-SV positive HCC. Niclosamide analogs improve upon the parental drug by overcoming its limited bioavailability and its dose limiting GI toxicity while shedding light on additional pathways for further improvement. Presentation: Saturday, June 17, 2023

B-003 Decrease in Serum apoE-rich HDL-cholesterol May Be Associated With Cholesterol Accumulation in the Liver of Rats Fed A High-cholesterol Diet

Abstract Background Free cholesterol accumulation in the liver has been suggested to be one of risk factors for the progression of non-alcoholic fatty liver to non-alcoholic steatohepatitis. Therefore, a non-invasive approach to assess hepatic cholesterol accumulation in the early stage without elevation of liver enzymes (AST and ALT) may be useful in clinical setting. In this study, we show that serum apoE-rich HDL-cholesterol level is a possible indicator for predicting hepatic cholesterol accumulation in rats with diet-induced simple liver steatosis. Methods Male Sprague-Dawley rats (8 weeks) were purchased from Charles River Laboratories. Four different diets were obtained from Oriental Yeast; control diet (MF), high-fat diet (HF; 68% MF, 30% palm oil, and 2% cholic acid), high-cholesterol diet (HC; 95.5% MF, 2.5% cholesterol, and 2% cholic acid), and high-fat/high-cholesterol diet (HFC; 81.75% MF, 15% palm oil, 1.25% cholesterol, and 2% cholic acid). The rats were randomly divided into 4 groups and fed as follows. The control and HFC groups received the MF and HFC diets for 8 weeks, respectively. The HF/HC group received the HF diet for the first 4 weeks and the HC diet for the next 4 weeks. The HC/HF group received the HC diet first, then the HF diet. Blood and liver tissue specimens were collected under anesthesia. Total HDL fractions were isolated from whole sera by the polyethylene glycol precipitation method, and applied into a cation-exchange column (HiTrap SPHP, GE healthcare) for determining apoE-rich HDL-cholesterol levels. Correlations between variables were assessed with Spearman’s correlation coefficient (rs). Results Total cholesterol content of the liver was significantly higher in the HFC (11.1-fold, P &amp;lt; 0.001), HF/HC (9.1-fold, P &amp;lt; 0.001), and HC/HF (5.8-fold, P &amp;lt; 0.001) groups than the control group. Hepatic free cholesterol was also higher in the HFC (5.5-fold, P &amp;lt; 0.001), HF/HC (3.9-fold, P &amp;lt; 0.001), and HC/HF (3.3-fold, P &amp;lt; 0.001) groups. Hepatic triglycerides were slightly increased in the HC/HF (1.6-fold, P &amp;lt; 0.01) and HFC (1.5-fold, P &amp;lt; 0.05) groups. Serum apoE-rich HDL-cholesterol was significantly decreased in the HF/HC (0.21-fold, P &amp;lt; 0.001), HC/HF (0.25-fold, P &amp;lt; 0.001) and HFC (0.42-fold, P &amp;lt; 0.01) groups. The HFC group had significantly higher serum ALT (3.0-fold, P &amp;lt; 0.05) and AST (2.3-fold, P &amp;lt; 0.01) levels, compared with the control groups. There was a significant increase in hepatic mRNA expression of TNF-α (24.8-fold, P &amp;lt; 0.01) and TGF-β (3.8-fold, P &amp;lt; 0.001) only in the HFC group, indicating liver inflammation and stellate cell activation. To exclude the effects of hepatocyte injury on apoE-rich HDL metabolism, rats with ALT less than 100 U/mL (n = 16) were included in the following analysis. ApoE-rich HDL-cholesterol levels were significantly and inversely correlated with hepatic total cholesterol (rs = −0.535, P &amp;lt; 0.05) and free cholesterol (rs = −0.532, P &amp;lt; 0.05) levels but not with hepatic triglycerides. Furthermore, the ratio of ALT to apoE-rich HDL-cholesterol was more highly correlated with hepatic total cholesterol (rs = 0.626, P &amp;lt; 0.01) and free cholesterol (rs = 0.632, P &amp;lt; 0.01). There was no such correlation for apoE-poor HDL-cholesterol. Conclusion: The present observations suggest that serum apoE-rich HDL-cholesterol levels may be a useful indicator for cholesterol accumulation in the liver of cholesterol-fed rats.

Abstract 4058: In vitro binding, efficacy and safety studies to support engineered T cell therapies

Abstract Engineered T cell therapies such as Chimeric Antigen Receptor (CAR) T cells and T cell receptor (TCR)-engineered T cells have emerged as a promising cancer therapy. To date, four anti-CD19 CAR-T and two anti-BCMA CAR-T products have been approved by the FDA for the treatment of hematological malignancies. Many more T cell therapy products are currently being explored, directed towards both liquid and solid tumors as well as for other clinical indications. High-quality and robust in vitro and in vivo assays are essential for the discovery and characterization of lead T cell therapy products. Furthermore, despite demonstrating therapeutically successful, the further development of T cell immunotherapies has been hindered by safety concerns. Selected target antigens might be expressed in healthy tissues or engineered T cells may non-specifically bind to antigens in healthy tissues, potentially resulting in severe side effects. In addition, the random integration of the CAR- or TCR-encoding DNA cassettes in the host cell genome has the potential risk of causing insertional mutagenesis and may contribute to oncogenic transformation of the T cells. The aim of this study was to develop several in vitro assays for the assessment of target cell binding, efficacy and safety of T cell therapies using CAR-T cells targeting the Human Epidermal growth factor Receptor 2 (HER2) as a model system. The z-Movi cell avidity analyzer was used to study the binding strength between the CAR-T cells and target tumor cells. Cytotoxicity co-culture assays were developed using increasing effector:target cells ratios and an impedance-based readout to quantify the viability of HER2-positive and -negative cancer cell lines in real-time, to confirm the activity and selectivity of the HER2-CAR-T cells. Furthermore, co-culture assays were also developed for a variety of primary or iPSC-derived healthy human cells (representing various tissues) to assess potential off-tumor effects of the HER2-CAR-T cells against healthy cells. In addition, an oncogenicity assay was developed to quantify the survival and proliferation of the HER2-CAR-T cells in the absence and presence of cytokines by flow cytometry, to determine whether the genomic editing of the T cells affected their cytokine-dependency. In conclusion, Charles River Laboratories developed several in vitro assays for the preclinical assessment of T cell therapy binding, efficacy, potency, specificity and safety to aid early-stage lead discovery, optimization and development, and to support Investigational New Drug (IND) applications. Charles River has the capabilities to support a full CAR/TCR program from inception to IND filing. Citation Format: Sophie Vermond, Benita Quist, Monique Hazenoot, Rene McLaughlin, David Cobeta Lopez, Namrata Jayanth, Folkert Verkaar, Omar Aziz, Maria LH Vlaming, Sabrina de Munnik, Gemma Moiset. In vitro binding, efficacy and safety studies to support engineered T cell therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4058.

The Next Generation of Drugs Will Be Enhanced by Machine Learning

Genetic Engineering & Biotechnology NewsVol. 43, No. 4 DepartmentsThe Next Generation of Drugs Will Be Enhanced by Machine LearningExperts from Charles River and Valo Health describe how artificial intelligence will change the drug discovery landscapeGrant Wishart and Guido LanzaGrant WishartGrant Wishart, PhD, is senior director, small molecule drug discovery and Logica lead at Charles River Laboratories. Website: www.criver.com. Guido Lanza is vice president of integrated research and Logica general manager at Valo Health. Website: www.valohealth.com.Search for more papers by this author and Guido LanzaGrant Wishart, PhD, is senior director, small molecule drug discovery and Logica lead at Charles River Laboratories. Website: www.criver.com. Guido Lanza is vice president of integrated research and Logica general manager at Valo Health. Website: www.valohealth.com.Search for more papers by this authorPublished Online:10 Apr 2023https://doi.org/10.1089/gen.43.04.06AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 43Issue 4Apr 2023 Information© 2023 by GEN PublishingTo cite this article:Grant Wishart and Guido Lanza.The Next Generation of Drugs Will Be Enhanced by Machine Learning.Genetic Engineering & Biotechnology News.Apr 2023.16-17, 19.http://doi.org/10.1089/gen.43.04.06Published in Volume: 43 Issue 4: April 10, 2023PDF download

Late Pregnant Dahl Salt-Sensitive Rats from Charles River Laboratories Do Not Exhibit Superimposed Preeclampsia.

Healthy pregnancy is characterized by a reduction in total peripheral vascular resistance which produces a decrease in maternal blood pressure. Failure of this normal maternal adaptation to pregnancy results in hypertensive disorders of pregnancy, which are dangerous for both the mother and fetus. Recently, it has been proposed that Dahl salt-sensitive (SS) rats are a model of spontaneous superimposed preeclampsia when maintained on a normal salt diet. Since these reports are from animals that are derived from sources that are not commercially available, the purpose of this study was to evaluate the blood pressure and pregnancy outcomes of SS rats from a commercial vendor. Mean arterial blood pressure was measured by indwelling femoral catheter in anesthetized SS virgin and SS day 21 late pregnant rats from Charles River Laboratories (CRL). Fetal outcomes from SS rats and control Sprague-Dawley (SD) rats were also measured. All rats were euthanized by exsanguination under anesthesia and tissues weighed. Virgin SS rats were found to have normal mean arterial blood pressure (102 ± 2mmHg), and late pregnant SS rats had the normal decrease in maternal blood pressure. SS rats were also found to have normal pregnancy outcomes. These results suggest that SS rats from CRL are not a model of superimposed preeclampsia. Further studies will be aimed at determining the differences between the blood pressure phenotype and pregnancy outcomes of existing colonies of SS rats in order to elucidate the mechanisms permitting the development of preeclampsia in certain colonies of this strain.

Lentiviral vector CDMO gets funding

iVexSol, a contract development and manufacturing organization (CDMO) that produces lentiviral vectors (LVVs), has raised $23.8 million in series A-3 financing. New investors Bristol Myers Squibb, Asahi Kasei Medical, and Charles River Laboratories joined existing investors to bring the total in series A fundng to over $39 million, according to the company. iVexSol says it will use the money to invest in its intelligent vector solutions by advancing technologies, growing technical teams, and building on current infrastructure “to support the production of stable LVV producer cell lines.”

The Role and underlying mechanism of dental pulp stem cell-derived exosomal miR-31 in the treatment of osteoarthritis by targeting mTOR to enhance chondrocyte autophagy levels

IntroductionOsteoarthritis is the most prevalent musculoskeletal progressive disease that leads to functional impairment and decreased quality of life. However, the current treatments remain unsatisfactory. Recent studies reveal that exosomes derived from mesenchymal stem cells offer a promising approach to improve the pathological changes of osteoarthritis, cartilage tissue, and chondrocyte homeostasis.Material and methodsA randomized, controlled animal experiment was conducted on nine-week-old male C57BL/6 mice that were obtained from Charles River Laboratories. In this in vitro and in vivo study, we studied the role and mechanisms of dental pulp stem cell-derived exosomes (DPSCs-exosomes) on osteoarthritis in the mouse model.ResultsThe study findings showed that a dental pulp stem cell could generate typical-characteristic exosomes. The injection of DPSCs-exosomes ameliorated destruction of cartilage, promoted matrix synthesis, inhibited cell apoptosis, and decreased the catabolic factors expression. However, this effect was shown to be almost eliminated when miR-31 antagomir was injected.ConclusionsFurthermore, DPSCs-exosomes show an ability to promote autophagy in chondrocytes through mTOR inhibition, in addition to reducing the mTOR luciferase activity. The ability of DPSCs-exosomes to partially regulate autophagy was blocked upon inhibition of miR-31. In brief, DPSCs-exosomes have a chondroprotective role in the mice osteoarthritis model. The underlying mechanism is possibly related to miR-31-mediated suppression of the mTOR-autophagy pathway.

Three Ways CRISPR Is Making Animal Research Models More Predictive

Genetic Engineering & Biotechnology NewsVol. 43, No. 1 TrendsThree Ways CRISPR Is Making Animal Research Models More PredictiveCharles River Laboratories describes how CRISPR-based animal models can help developers improve target engagement, human efficacy, and safetyLieke Geerts, PhDLieke Geerts, PhDLieke Geerts, PhD, is a group leader at Charles River Laboratories. Website: www.criver.com.Search for more papers by this authorPublished Online:11 Jan 2023https://doi.org/10.1089/gen.43.01.14AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 43Issue 1Jan 2023 Information© 2023 by GEN PublishingTo cite this article:Lieke Geerts, PhD.Three Ways CRISPR Is Making Animal Research Models More Predictive.Genetic Engineering & Biotechnology News.Jan 2023.40-42.http://doi.org/10.1089/gen.43.01.14Published in Volume: 43 Issue 1: January 11, 2023PDF download

1571. Hospital COVID-19 Burden Impact on Inpatient Antibiotic Use Rates

Abstract Background COVID-19 shifted antibiotic stewardship program resources and changed antibiotic use (AU). Shifts in patient populations with COVID surges, including pauses to surgical procedures, and dynamic practice changes makes temporal associations difficult to interpret. Our analysis aimed to address the impact of COVID on AU after adjusting for other practice shifts. Methods We performed a longitudinal analysis of AU data from 30 Southeast US hospitals. Three pandemic phases (1: 3/20–6/20; 2: 7/20–10/20; 3: 11/20–2/21) were compared to baseline (1/2018–1/2020). AU (days of therapy (DOT)/1000 patient days (PD)) was collected for all antimicrobial agents and specific subgroups: broad spectrum (NHSN group for hospital-onset infections), CAP (ceftriaxone, azithromycin, levofloxacin, moxifloxacin, and doxycycline), and antifungal. Monthly COVID burden was defined as all PD attributed to a COVID admission. We fit negative binomial GEE models to AU including phase and interaction terms between COVID burden and phase to test the hypothesis that AU changes during the phases were related to COVID burden. Models included adjustment for Charlson comorbidity, surgical volume, time since 12/2017 and seasonality. Results Observed AU rates by subgroup varied over time; peaks were observed for different subgroups during distinct pandemic phases (Figure). Compared to baseline, we observed a significant increase in overall, broad spectrum, and CAP groups during phase 1 (Table). In phase 2, overall and CAP AU was significantly higher than baseline, but in phase 3, AU was similar to baseline. These phase changes were separate from effects of COVID burden, except in phase 1 where we observed significant effects on antifungal (increased) and CAP (decreased) AU (Table). Conclusion Changes in hospital AU observed during early phases of the COVID pandemic appeared unrelated to COVID burden and may have been due to indirect pandemic effects (e.g., case mix, healthcare resource shifts). By pandemic phase 3, these disruptive effects were not as apparent, potentially related to shifts in non-COVID patient populations or ASP resources, availability of COVID treatments, or increased learning, diagnostic certainty, and provider comfort with avoiding antibacterials in patients with suspected COVID over time. Disclosures Melissa D. Johnson, PharmD, Biomeme: Licensed Transcriptional Signature for Candidemia|Charles River Laboratories: Grant/Research Support|Entasis Therapeutics: Advisor/Consultant|Merck &amp; Co. Inc: Advisor/Consultant|Merck &amp; Co. Inc: Grant/Research Support|Pfizer, Inc.: Advisor/Consultant|Scynexis Inc.: Grant/Research Support|Theratechnologies: Advisor/Consultant Angelina Davis, PharmD, M.S., Merck &amp; Co.: Honoraria Sonali D. Advani, MBBS, MPH, FIDSA, Locus Biosciences: Advisor/Consultant|Locus Biosciences: Honoraria|Sysmex America: Advisor/Consultant Ibukun Kalu, MD, Pfizer, Inc.: Institutional support for clinical trial Rebekah W. Moehring, MD, MPH, FIDSA, FSHEA, UpToDate, Inc.: Author Royalties.

1570. Cumulative Antibiotic Exposure and Risk for Candidemia

Abstract Background Broad-spectrum antibiotic use is a known risk factor for candidemia, but the duration and type of antibiotic exposure associated with greatest risk are not well characterized. Measurements of antibiotic days may be useful to assess cumulative burden of selective pressure on the microbiome and risk for candidemia. Methods This retrospective cohort study aimed to quantify the effect of antibiotic exposure on risk for candidemia. The primary outcome was hazard for developing candidemia across an array of antibiotic agents, classes and spectra. We measured antibiotic days of therapy (DOT) for adults admitted to Duke University hospitals 1/1/2016–12/31/2021. We excluded patients with community-onset candidemia, defined as growth of Candida spp. in blood culture collected ≤ 48 hours of admission, because antibiotic exposure prior to arrival was not reliably accessible. Time-to-event analyses were performed using the Nelson-Aalen estimator for modeling cumulative hazard functions to compare the proportion of candidemia observed based on exposure to each antibiotic. Logrank tests were used to evaluate for differences between hazard functions with a pre-specified alpha level of 0.05, and exponential cumulative proportional hazards models were implemented to generalize hazard functions. Results During 164,185 encounters in 105,330 unique patients, we identified candidemia in 237 patients. Prior to developing candidemia, cases received a total of 9,604 antibacterial DOT distributed across 46 unique antibiotic agents (Fig. 1) Carbapenems were associated with increased hazard for candidemia compared to beta-lactams (p&amp;lt; 0.005) (Figs. 2–3). There were 57 encounters for candidemia where meropenem was administered with a median of 10 DOT prior to onset of candidemia. Days of Antibiotic Therapy by Agent and Outcome. Conclusion This work represents a novel approach to quantifying antibiotic exposure as a risk factor for candidemia. In an unadjusted model, we identified carbapenems as a high-risk class; additional analysis with adjusted regression models will help contextualize exposure risk with respect to comorbidity and illness severity. This work may serve as a reference for antibiotic stewards as they promote appropriate antibiotic use, including reducing overuse of broad-spectrum antibiotics. Disclosures Rebekah W. Moehring, MD, MPH, FIDSA, FSHEA, UpToDate, Inc.: Author Royalties Barbara D. Alexander, MD, Astellas: Advisor/Consultant|HealthtrackRx: Advisor/Consultant|HealthtrackRx: Grant/Research Support|Scynexis: Grant/Research Support|UpToDate: Advisor/Consultant Melissa D. Johnson, PharmD, Biomeme: Licensed Transcriptional Signature for Candidemia|Charles River Laboratories: Grant/Research Support|Entasis Therapeutics: Advisor/Consultant|Merck &amp; Co. Inc: Advisor/Consultant|Merck &amp; Co. Inc: Grant/Research Support|Pfizer, Inc.: Advisor/Consultant|Scynexis Inc.: Grant/Research Support|Theratechnologies: Advisor/Consultant.

791. ICU Provider Overestimation of Ventilator-Associated Pneumonia Diagnostic Probability: An Opportunity for Diagnostic Stewardship

Abstract Background Overdiagnosis of ventilator-associated pneumonia (VAP) is common in intensive care units (ICUs). Because VAP is a clinical diagnosis without an easily accessible confirmatory diagnostic test, understanding how ICU physicians practice diagnostic reasoning in suspected VAP is vital to informing diagnostic stewardship efforts. We aimed to assess the accuracy of ICU practitioner estimates of VAP diagnostic probability. Methods We conducted a survey of physicians at Michigan Medicine ICUs. Providers were presented with clinical vignettes (Figure 1) in which mechanically ventilated patients developed clinical signs suggestive of VAP and then had radiographic/microbiologic tests performed. Providers were asked to estimate the probability of VAP before and after each presented clinical sign or test result, which were then used to impute provider-estimated likelihood ratios (pLRs) for each clinical sign and diagnostic test. Provider-estimated probabilities and LRs were compared to evidence-based VAP probabilities and LRs (eLRs) derived from systematic reviews and meta-analyses. Survey Case Example. Results Of 102 survey respondents, 54% felt moderately to extremely confident in their ability to accurately diagnose VAP. Providers significantly overestimated the pre and post-test probability of VAP in all clinical scenarios (provider-estimated VAP diagnostic probabilities are visualized using density plots relative to evidence-based diagnostic probability in Figure 2). Median pLRs for nearly all VAP clinical signs and diagnostic tests were also significantly higher than eLRs (Figure 3), most notably for isolated purulent endotracheal secretions (pLR 1.54 vs eLR 0.76, p&amp;lt; 0.01), positive chest radiograph (pLR 6.0 vs eLR 3.55, p&amp;lt; 0.01), and positive bronchoalveolar lavage culture (pLR 5.7 vs eLR 1.37, p&amp;lt; 0.01). Density Plots of Provider-Estimated vs Evidence-Based Diagnostic Probability of Ventilator-Associated Pneumonia. Provider-Estimated vs Evidence-Based Diagnostic Likelihood Ratios for Clinical Signs and Test Results Related to Ventilator-Associated Pneumonia. Conclusion Physicians routinely overestimated the diagnostic probability of VAP and the positive LRs of possible VAP clinical signs, as well as VAP diagnostic radiographic and microbiologic tests. Educational initiatives addressing and calibrating ICU providers’ perceptions of VAP diagnostic probability warrant investigation as an antimicrobial stewardship tool. Disclosures Owen Albin, MD, Charles River Laboratory: Advisor/Consultant|Cipla Pharmaceuticals: Advisor/Consultant|Shionogi Inc: Advisor/Consultant.

2019. Understanding the Impact of COVID-19 Pandemic on Central Line-Associated Bloodstream Infections (CLABSIs): Expanding Analysis to the Microbiologic Level

Abstract Background Increases in central line-associated bloodstream infection (CLABSI) rates have been reported in association with the COVID-19 pandemic, particularly among Candida species and coagulase-negative Staphylococcal species (CoNS). We sought to further validate the impact of the COVID-19 pandemic on CLABSI trends and perform a microbiologic analysis. Methods This is an IRB-approved retrospective analysis of CLABSIs across a network of 38 community hospitals in southeastern United States. CLABSI rates were compared between pre-pandemic (1/1/2018-3/30/2020) and pandemic periods (4/1/2020-12/31/2021). Regression models were developed to evaluate CLABSI incidence over time. Likelihood ratio tests were used to compare models that were exclusively time-dependent to segmented regression models that also accounted for the COVID-19 pandemic. Results A total of 1,167 CLABSIs over 1,345,062 central line days were analyzed (Table 1). The mean monthly CLABSI rate per hospital increased from 0.63 to 1.01 per 1,000 central line days (p&amp;lt; 0.001) in the pandemic period (Table 1). CLABSIs secondary to Candida (0.16 to 0.33, p&amp;lt; 0.001), CoNS (0.09 to 0.22, p&amp;lt; 0.001), and Enterococcal species (0.06 to 0.18, p=0.001) increased, while Escherichia coli CLABSIs decreased (0.04 to 0.01, p&amp;lt; 0.001). Upon regression modeling, the COVID-19 pandemic was associated with increases in monthly CLABSI rates by Candida and Enterococcus species (Figure 1). In contrast, the changes in CoNS and Escherichia coli CLABSI rates were better explained by exclusively time-dependent models (Figure 1; Table 2). Non-sustained changes in Staphylococcus aureus and Klebsiella pneumoniae CLABSI rates were also noted (Table 2). Table 1:Count data, central line days, and mean monthly CLABSI incidence by organism per hospital. Rates are provided as CLABSIs per 1,000 central line days. Figure 1:Regression analysis of monthly CLABSI rates by pathogen. Gray areas denote COVID-19 pandemic period. Statistically significant level changes in CLABSI rates were observed among Candida and Enterococcus spp. (RR=1.92, CI 1.16-3.20 and 2.42, CI 1.09-5.38). Staphylococcus aureus CLABSI rates had a non-sustained but significant increase at the onset of COVID-19 (RR 2.20, CI 1.16-4.20). CoNS and E. coli rate changes occurred independent of COVID-19 (see Table 2). Table 2:Coefficient table of regression analyses for CLABSI rates by pathogen. Exclusively time-dependent models were compared to segmented regression models for each organism and, if no significant difference was noted between models, only the time-dependent model was applied. Conclusion The COVID-19 pandemic was associated with substantial increases in CLABSIs, driven in part by Candida and Enterococcus species across this network of hospitals. However, the observed increase in CoNS CLABSIs and decrease in Escherichia coli CLABSIs appear to have occurred independently of COVID-19, which only became apparent upon regression analysis. Interpretation of pre-post statistics without assessment of pre-existing trends should be done cautiously. Additional analyses may help elucidate other factors influencing these CLABSI trends by organism. Disclosures Sonali D. Advani, MBBS, MPH, FIDSA, Locus Biosciences: Advisor/Consultant|Locus Biosciences: Honoraria|Sysmex America: Advisor/Consultant Melissa D. Johnson, PharmD, MHS, AAHIVP, Charles River Laboratories: Grant/Research Support|Entasis: Honoraria|Merck: Grant/Research Support|Pfizer: Honoraria|Scynexis: Grant/Research Support|Theratechnologies: Honoraria|UpToDate: Honoraria.

424. Risk Factors for Infection or Colonization with Piperacillin-tazobactam (TZP) Resistant P. aeruginosa (PSA) with Increasing Degrees of β-lactam Resistance

Abstract Background Resistance to β-lactams amongst PSA is common with roughly 20% of isolates demonstrating resistance to TZP. This can lead to delays in the time to appropriate empiric therapy. While rapid diagnostics can identify PSA at earlier time points, they fail to detect common β-lactam resistance mechanisms in this pathogen. Though risk factors for TZP resistance (TZP-R) amongst PSA have been identified, co-resistance amongst traditional anti-PSA β-lactams is common, so decisions to turn to other traditional anti-PSA or novel agents in the presence of such predictors are unclear. This study aims to find predictors unique to varying degrees of β-lactam resistance amongst TZP-R PSA to inform these decisions. Methods This was a retrospective case-case-case-control study of all non-cystic fibrosis patients with PSA isolated from a culture from 2015-2021 at Michigan Medicine. Four study groups were created based on the susceptibility profile of PSA: TZP susceptible (TZP-S), resistance to TZP only (TZP-R ONLY), resistance to TZP in addition to 1 or 2 other traditional anti-PSA β-lactams (TZP-R PLUS), and resistance to all traditional anti-PSA β-lactams (pan β-lactam resistance, PBR). Bivariate and multivariate analyses were performed to compare each degree of TZP resistance to the TZP-S group. Results were then compared across models to assess uniqueness to a given resistant phenotype. Results 2365 patients were included, and results of the multivariate analyses are listed in the table. The sole predictor of TZP-R ONLY was recent ceftriaxone or cefotaxime use. Independent predictors unique to TZP-R PLUS were community acquired infection, recent ICU residence, and recent exposure to TZP or aztreonam. Most risk factors identified were either unique to PBR or shared between PBR and TZP-R PLUS including history of PSA, history of TZP-R PSA, and recent exposure to multiple antibiotics. Multivariate Models for TZP-R ONLY, TZP-R PLUS, and PBR Conclusion This analysis demonstrates that previously identified risk factors for TZP-R P. aeruginosa are predictors of PSA with greater degrees of resistance. Given these findings, consideration needs to be given to escalation to novel agents in the presence of risk factors at the time of PSA isolation, rather than to other traditional anti-PSA β-lactams. Disclosures Owen Albin, MD, Charles River Laboratory: Advisor/Consultant|Cipla Pharmaceuticals: Advisor/Consultant|Shionogi Inc: Advisor/Consultant Keith S. Kaye, MD, GSK: Advisor/Consultant|merck: Advisor/Consultant|merck: Stocks/Bonds|Qpex: Advisor/Consultant|Shionogi: Advisor/Consultant|Spero: Advisor/Consultant jason M. Pogue, PharmD, consultant to Venatorx, Merck, Shionogi, QPex Biopharma, Utility, GSK, and Entasis.: Advisor/Consultant.