Common human genetic variants of APOE impact murine COVID-19 mortality.

Clinical outcomes of severe acute respiratory syndrome 2 (SARS-CoV-2) infection are highly heterogeneous, ranging from asymptomatic infection to lethal coronavirus disease 2019 (COVID-19). The factors underlying this heterogeneity remain insufficiently understood. Genetic association studies have suggested that genetic variants contribute to the heterogeneity of COVID-19 outcomes, but the underlying potential causal mechanisms are insufficiently understood. Here we show that common variants of the apolipoprotein E (APOE) gene, homozygous in approximately 3% of the world's population1 and associated with Alzheimer's disease, atherosclerosis and anti-tumour immunity2-5, affect COVID-19 outcome in a mouse model that recapitulates increased susceptibility conferred by male sex and advanced age. Mice bearing the APOE2 or APOE4 variant exhibited rapid disease progression and poor survival outcomes relative to mice bearing the most prevalent APOE3 allele. APOE2 and APOE4 mice exhibited increased viral loads as well as suppressed adaptive immune responses early after infection. In vitro assays demonstrated increased infection in the presence of APOE2 and APOE4 relative to APOE3, indicating that differential outcomes are mediated by differential effects of APOE variants on both viral infection and antiviral immunity. Consistent with these in vivo findings in mice, our results also show that APOE genotype is associated with survival in patients infected with SARS-CoV-2 in the UK Biobank (candidate variant analysis, P = 2.6 × 10-7). Our findings suggest APOE genotype to partially explain the heterogeneity of COVID-19 outcomes and warrant prospective studies to assess APOE genotyping as a means of identifying patients at high risk for adverse outcomes.

Chlamydia muridarum infection causes bronchointerstitial pneumonia in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice.

The murine bacterial pathogen Chlamydia muridarum (Cm) has been used to study human Chlamydia infections in various mouse models. CD4+ T-cells, natural killer cells, and interferon-gamma (IFN-γ)-mediated immunity are important to control experimentally induced Cm infections. Despite its experimental use, natural infection by Cm has not been documented in laboratory mice since the 1940s. In 2022, the authors reported the discovery of natural Cm infections in numerous academic institutional laboratory mouse colonies around the globe. To evaluate the impact of Cm infection in severely immunocompromised mice, 19 NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were cohoused with Cm shedding, naturally infected immunocompetent mice and/or their soiled bedding for 4 weeks and subsequently euthanized. Clinical disease, characterized by lethargy, dyspnea, and weight loss, was observed in 11/19 NSG mice, and 16/18 NSG mice had neutrophilia. All mice exhibited multifocal to coalescing histiocytic and neutrophilic bronchointerstitial pneumonia (17/19) or bronchiolitis (2/19) with intraepithelial chlamydial inclusions (CIs). Immunofluorescence showed CIs were often associated with bronchiolar epithelium. CIs were frequently detected by immunohistochemistry in tracheal and bronchiolar epithelium (19/19), as well as throughout the small and large intestinal epithelium without lesions (19/19). In a subset of cases, Cm colonized the surface epithelium in the nasopharynx (16/19), nasal cavity (7/19), and middle ear canal (5/19). Endometritis and salpingitis with intraepithelial CI were identified in a single mouse. These findings demonstrate that Cm infection acquired through direct contact or soiled bedding causes significant pulmonary pathology and widespread intestinal colonization in NSG mice.

Subcutaneous choriocarcinomas in captive Amargosa voles (Microtus californicus scirpensis).

Spontaneous choriocarcinomas are rare, highly vascular, malignant trophoblastic tumors that occur in humans and animals. This report describes the unusual spontaneous presentation of 4 choriocarcinomas within the subcutaneous tissues of 4, multiparous but nongravid, Amargosa voles (Microtus californicus scirpensis) from a captive breeding colony. Two subcutaneous neoplasms were composed of multifocal discohesive and infiltrative aggregates of medium to large trophoblasts and cytotrophoblasts within a fibrovascular stroma. Neoplastic cells were associated with variably sized thrombi and cavitary areas of hemorrhage and necrosis. Two subcutaneous tumors were predominantly composed of expansile, blood-filled, cystic spaces lined by neoplastic cytotrophoblasts and occasionally contained medium to large trophoblasts. Trophoblasts and cytotrophoblasts were positive for pancytokeratin and cytokeratin 8/18, negative for alpha-fetoprotein, and contained intracytoplasmic Periodic acid-Schiff (PAS)-positive glycogen in all 4 tumors. In species with hemochorial placentation, migration of trophoblasts into maternal circulation with embolization to distant nonreproductive tissues occurs and may explain the unusual subcutaneous distribution of these 4 tumors. The 2 multiloculated paucicellular tumors may represent an early stage of neoplastic transformation. To the authors' knowledge, this is the first report characterizing choriocarcinomas in extrareproductive sites in rodents.

Anaplastic nephroblastoma with peritoneal metastasis in an adult female Sprague Dawley rat.

Spontaneous nephroblastomas are uncommon tumors of laboratory rats. This report describes a spontaneous nephroblastoma with peritoneal metastasis in an 11-month-old, female Sprague Dawley rat. The rat was part of a breeding program and presented 15 days post parturition with clinical signs including tachypnea, dyspnea and abdominal distension. At necropsy, the right kidney was markedly enlarged by an expansile pale-tan to white multinodular mass with extension into the retroperitoneal space, with multifocal variably sized nodules involving the mesentery, and surface of pancreas, liver, uterus, and ovarian bursa. The rat also had severe bicavitary effusion. Histologically, the renal parenchyma of the affected kidney was replaced by a moderately cellular, poorly-demarcated, non-encapsulated, multilobulated mass that appeared to compress the adjacent renal outer medulla and cortex. Three distinct neoplastic cell populations were identified in this renal tumor: epithelial cells (convoluted and dilated tubules / rare primitive glomeruloid structures), mesenchymal (neoplastic spindle cells in connective tissue), and blastemal cells (primitive neoplastic cells). The extrarenal nodular masses were predominantly composed of neoplastic mesenchymal and pleomorphic blastemal cells. Immunohistochemically, neoplastic epithelial cells in the renal mass were positive for pancytokeratin, and blastemal cells in both renal and extrarenal masses were positive for Wilms' tumor 1 protein (WT1) and vimentin. Neoplastic mesenchymal elements in both renal and extrarenal masses were positive for vimentin. The neoplasm was negative for chromogranin A and S100. The tumor was classified as an anaplastic nephroblastoma with metastasis to the mesentery and peritoneal organs.

Traumatic phacocele in an American Miniature Horse.

An 18-year-old American Miniature Horse mare was presented with a complaint of a scleral swelling affecting the right eye and a history of suspected trauma 6 weeks prior to evaluation. Clinical findings included severe blepharospasm, a bulbous swelling of the dorsotemporal bulbar conjunctiva, and phthisis bulbi. Ocular ultrasound was recommended but declined. Enucleation was elected for the blind, painful eye and was performed standing. Gross and histopathologic examinations of the globe were consistent with extrusion of the lens to the episcleral space, which is classified as a traumatic phacocele when associated with naturally occurring trauma. The location of lens entrapment suggested globe rupture occurred at the limbus, which is described as one of the weakest points of the equine globe. Subconjunctival dislocation of the lens and development of a traumatic phacocele should be considered as a differential diagnosis for horses presenting with subconjunctival masses, apparent aphakia, and historical trauma.

Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, encounters two disparate host environments during its enzootic life cycle, Ixodes ticks and mammalian hosts. B. burgdorferi has a small genome that encodes a streamlined cyclic dimeric GMP (c-di-GMP) signaling system comprising a single diguanylate cyclase, Rrp1, and two phosphodiesterases. This system is essential for spirochete survival in ticks, in part because it controls the expression of the glp operon involved in glycerol utilization. In this study, we showed that a B. burgdorferi c-di-GMP receptor, PlzA, functions as both a positive and a negative regulator for glp expression. Deletion of plzA or mutation in plzA that impaired c-di-GMP binding abolished glp expression. On the other hand, overexpression of plzA resulted in glp repression, which could be rescued by simultaneous overexpression of rrp1. plzA overexpression in the rrp1 mutant, which is devoid of c-di-GMP, or overexpression of a plzA mutant incapable of c-di-GMP binding further enhanced glp repression. Combined results suggest that c-di-GMP-bound PlzA functions as a positive regulator, whereas ligand-free PlzA acts as a negative regulator for glp expression. Thus, PlzA of B. burgdorferi with a streamlined c-di-GMP signaling system not only controls multiple targets, as previously envisioned, but has also evolved different modes of action.IMPORTANCE The Lyme disease pathogen, Borrelia burgdorferi, has a simple cyclic dimeric GMP (c-di-GMP) signaling system essential for adaptation of the pathogen to the complicated tick environment. The c-di-GMP effector of B. burgdorferi, PlzA, has been shown to regulate multiple cellular processes, including motility, osmolality sensing, and nutrient utilization. The findings of this study demonstrate that PlzA not only controls multiple targets but also has different functional modalities, allowing it to act as both positive and negative regulator of the glp operon expression. This work highlights how bacteria with a small genome can compensate for the limited regulatory repertoire by increasing the complexity of targets and modes of action in their regulatory proteins.

Outer surface protein OspC is an antiphagocytic factor that protects Borrelia burgdorferi from phagocytosis by macrophages.

Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface of Borrelia burgdorferi during tick feeding and the early phase of mammalian infection. OspC is required for B. burgdorferi to establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses. However, the exact biological function of OspC remains elusive. In this study, we showed that the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγ(null) mice that lack B cells, T cells, NK cells, and lytic complement. The ospC mutant also could not establish infection in anti-Ly6G-treated SCID and C3H/HeN mice (depletion of neutrophils). However, depletion of mononuclear phagocytes at the skin site of inoculation in SCID and C3H/HeN mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted mice, the ospC mutant was able to colonize the joints and triggered neutrophilia during dissemination. Furthermore, we found that phagocytosis of green fluorescent protein (GFP)-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 macrophage-like cells, but not in PMN-HL60, was significantly higher than parental wild-type B. burgdorferi strains, suggesting that OspC has an antiphagocytic property. In addition, overproduction of OspC in spirochetes also decreased the uptake of spirochetes by murine peritoneal macrophages. Together, our findings provide evidence that mononuclear phagocytes play a key role in clearance of the ospC mutant and that OspC promotes spirochetes' evasion of macrophages during early Lyme borreliosis.

Chlamydia muridarum Associated Pulmonary and Urogenital Disease and Pathology in a Colony of Enzootically Infected Il12rb2 Deficient and Stat1 Knockout Mice.

Chlamydia muridarum (Cm), an intracellular bacterium of historical importance, was recently rediscovered as moderately prevalent in research mouse colonies. Cm was first reported as a causative agent of severe pneumonia in mice about 80 y ago, and while it has been used experimentally to model Chlamydia trachomatis infection of humans, there have been no further reports of clinical disease associated with natural infection. We observed clinical disease and pathology in 2 genetically engi- neered mouse (GEM) strains, Il12rb2 KO and STAT1 KO, with impaired interferon-γ signaling and Th1 CD4+ T cell responses in a colony of various GEM strains known to be colonized with and shedding Cm. Clinical signs included poor condition, hunched posture, and poor fecundity. Histopathology revealed disseminated Cm with lesions in pulmonary, gastrointestinal, and urogenital tissues. The presence of Cm was confirmed using both immunohistochemistry for Cm major outer membrane protein-1 antigen and in situ hybridization using a target probe directed against select regions of Cm strain Nigg. Cm was also found in association with a urothelial papilloma in one mouse. These cases provide additional support for excluding Cm from research mouse colonies.

Chlamydia muridarum Causes Persistent Subclinical Infection and Elicits Innate and Adaptive Immune Responses in C57BL/6J, BALB/cJ and J:ARC(S) Mice Following Exposure to Shedding Mice.

Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its' experimental use, there are limited studies evaluating Cm's effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naïve C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was found in numerous infected mice. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). These results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric infections with Cm which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.

Assessment of Antimicrobial Therapy in Eradicating Chlamydia muridarum in Research Mice: Immune Status and its Impact on Outcomes.

Chlamydia muridarum (Cm) is a moderately prevalent, gram-negative, intracellular bacterium that affects laboratory mice, causing subclinical to severe disease, depending on the host's immune status. The effectiveness of various antibiotic regimens aimed at eradicating Cm in both immunodeficient and immunocompetent laboratory mice was evaluated. NSG mice were cohoused with Cm-shedding BALB/cJ mice for 14 days to simulate natural exposure. Four groups of 8 infected NSG mice were treated for 7 days with either 0.08% sulfamethoxazole and 0.016% trimethoprim (TMS) in water, 0.0625% doxycycline in feed, 0.124%/0.025% TMS in feed, or 0.12% amoxicillin in feed. A control group was provided standard water and feed. The impact of treatment on gastrointestinal microbiota (GM) was performed through shotgun sequencing on the last day of treatment. TMS and Amoxicillin had negligible effects on GM, while doxycycline had the largest effect. All antibiotic treated NSG mice exhibited clinical disease, including dehydration, hunched posture, >20% weight loss, and dyspnea, leading to euthanasia 21-40 days post-treatment (32.6 ± 4.2 days; mean ± SD). Untreated controls were euthanized 14-33 days post-exposure (23.75 ± 5.9 days). All mice were fecal PCR positive for Cm at euthanasia. Histological evaluation revealed multifocal histiocytic and neutrophilic bronchointerstitial pneumonia and/or bronchiolitis featuring prominent intralesional chlamydial inclusion bodies in all mice. Subsequently, groups of 8 C57BL/6J, BALB/cJ, NOD.SCID, and NSG mice infected with Cm were treated with 0.124%/0.025% TMS in feed for 7 (BALB/cJ and C57BL/6J) or 21 days (NSG and NOD.SCID). All immunocompetent and NOD.SCID mice were negative for Cm by PCR 14 days post-treatment, remained clinically normal and had no evidence of Cm infection at necropsy, all NSG mice remained Cm positive and were euthanized. While these findings highlight the difficulties in eradicating Cm from highly immunodeficient mice, eradication of Cm from immunocompetent or moderately immunocompromised mice with antibiotics is feasible.

Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

IMPORTANCE: H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction.

Senescent cells, which accumulate in organisms over time, contribute to age-related tissue decline. Genetic ablation of senescent cells can ameliorate various age-related pathologies, including metabolic dysfunction and decreased physical fitness. While small-molecule drugs that eliminate senescent cells ('senolytics') partially replicate these phenotypes, they require continuous administration. We have developed a senolytic therapy based on chimeric antigen receptor (CAR) T cells targeting the senescence-associated protein urokinase plasminogen activator receptor (uPAR), and we previously showed these can safely eliminate senescent cells in young animals. We now show that uPAR-positive senescent cells accumulate during aging and that they can be safely targeted with senolytic CAR T cells. Treatment with anti-uPAR CAR T cells improves exercise capacity in physiological aging, and it ameliorates metabolic dysfunction (for example, improving glucose tolerance) in aged mice and in mice on a high-fat diet. Importantly, a single administration of these senolytic CAR T cells is sufficient to achieve long-term therapeutic and preventive effects.

Aging limits stemness and tumorigenesis in the lung by reprogramming iron homeostasis.

Aging is associated with a decline in the number and fitness of adult stem cells 1-4 . Aging-associated loss of stemness is posited to suppress tumorigenesis 5,6 , but this hypothesis has not been tested in vivo . Here, using physiologically aged autochthonous genetically engineered mouse models and primary cells 7,8 , we demonstrate aging suppresses lung cancer initiation and progression by degrading stemness of the alveolar cell of origin. This phenotype is underpinned by aging-associated induction of the transcription factor NUPR1 and its downstream target lipocalin-2 in the cell of origin in mice and humans, leading to a functional iron insufficiency in the aged cells. Genetic inactivation of the NUPR1-lipocalin-2 axis or iron supplementation rescue stemness and promote tumorigenic potential of aged alveolar cells. Conversely, targeting the NUPR1- lipocalin-2 axis is detrimental to young alveolar cells via induction of ferroptosis. We find that aging-associated DNA hypomethylation at specific enhancer sites associates with elevated NUPR1 expression, which is recapitulated in young alveolar cells by inhibition of DNA methylation. We uncover that aging drives a functional iron insufficiency, which leads to loss of stemness and tumorigenesis, but promotes resistance to ferroptosis. These findings have significant implications for the therapeutic modulation of cellular iron homeostasis in regenerative medicine and in cancer prevention. Furthermore, our findings are consistent with a model whereby most human cancers initiate in young individuals, revealing a critical window for such cancer prevention efforts.

Manganese and zinc regulate virulence determinants in Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection. B. burgdorferi is known to be unique in metal utilization, and little is known of the role of biologically available metals in B. burgdorferi. Here, we identified two transition metal ions, manganese (Mn(2+)) and zinc (Zn(2+)), that influenced regulation of RpoS. The intracellular Mn(2+) level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn(2+) repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn(2+). Conversely, a decrease of intracellular Mn(2+) by deletion of the Mn(2+) transporter gene, bmtA, resulted in elevated levels of RpoS and OspC. Mn(2+) affected RpoS through BosR, a Fur family homolog that is required for rpoS expression: elevated intracellular Mn(2+) levels greatly reduced the level of BosR protein but not the level of bosR mRNA. Thus, Mn(2+) and Zn(2+) appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn(2+) and Zn(2+) play a critical role in regulation of virulence in bacteria.

Examination of Horizontal Transmission of Nippostrongylus brasiliensis in Mice to Assess Biosecurity Risks.

Mice are commonly infected with Nippostrongylus brasiliensis (Nb) to study their immune responses. However, biosecurity measures have not been established for housing Nb-infected mice and rats. Transmission reportedly does not occur when infected mice are cohoused with naive mice. To test this, we inoculated female NOD. Cg-Prkdcscid Il2rgtm1Wjl /Sz(NSG;n = 12) and C57BL/6J (B6;n = 12) mice with 750 Nb L3 larvae. These mice were then cohoused with naïve NSG ( n = 24) and B6 ( n = 24) mice (1 infected and 2 naïve mice per cage (24 cages) for 28 d in static microisolation cages that were changed every 14 d. We also did several studies to determine the conditions that favor horizontal transmission. First, we assessed in vitro development to the L3 stage of Nb egg-containing fecal pellets maintained under 4 environmental conditions (dry, moist, soiled bedding, and control). Second, we assessed infection of naïve NSG mice ( n = 9) housed in microisolation cages that contained soiled bedding spiked with infective L3 larvae (10,000/cage). Third, we gavaged NSG mice ( n = 3) with Nb eggs to model the potential for infection after coprophagy. We found that naïve NSG (9 of 24) and B6 (10 of 24) mice cohoused with an infected cagemate passed Nb eggs in feces as early as 1 d after cohousing and intermittently thereafter for varying periods. This shedding was presumably the result of coprophagy because adult worms were not detected in the shedding mice at euthanasia. Although eggs developed in vitro into L3 larvae under moist and control environmental conditions, none of the NSG mice housed in cages with L3 -spiked bedding or gavaged with eggs became infected with Nb. These findings indicate that infectious horizontal transmission does not occur when mice are housed with Nb-shedding cage mates in static microisolation cages with a 14-d cage-changing interval. Results from this study can be used to inform biosecurity practices when working with Nb-infected mice.

Molecular origins of mutational spectra produced by the environmental carcinogen N-nitrosodimethylamine and SN1 chemotherapeutic agents.

DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O 6-methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of ∼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC→AT mutations in 5'-Pu-G-3' contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.

Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction.

Senescent cells accumulate in organisms over time because of tissue damage and impaired immune surveillance and contribute to age-related tissue decline1,2. In agreement, genetic ablation studies reveal that elimination of senescent cells from aged tissues can ameliorate various age-related pathologies, including metabolic dysfunction and decreased physical fitness3-7. While small-molecule drugs capable of eliminating senescent cells (known as 'senolytics') partially replicate these phenotypes, many have undefined mechanisms of action and all require continuous administration to be effective. As an alternative approach, we have developed a cell-based senolytic therapy based on chimeric antigen receptor (CAR) T cells targeting uPAR, a cell-surface protein upregulated on senescent cells, and previously showed these can safely and efficiently eliminate senescent cells in young animals and reverse liver fibrosis8. We now show that uPAR-positive senescent cells accumulate during physiological aging and that they can be safely targeted with senolytic CAR T cells. Treatment with anti uPAR CAR T cells ameliorates metabolic dysfunction by improving glucose tolerance and exercise capacity in physiological aging as well as in a model of metabolic syndrome. Importantly, a single administration of a low dose of these senolytic CAR T cells is sufficient to achieve long-term therapeutic and preventive effects.

Factors Affecting Hematologic and Serum Biochemical Parameters in Healthy Common Marmosets (Callithrix jacchus).

Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.

The Epidemiology of Invasive, Multipleantibiotic-resistant Klebsiella pneumoniae Infection in a Breeding Colony of Immunocompromised NSG Mice.

Klebsiella pneumoniae (Kp) is a gram-negative opportunistic pathogen that causes severe pneumonia, pyelonephritis, and sepsis in immunocompromised hosts. During a 4-mo interval, several NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) breeders and pups in our facilities were diagnosed with Kp infections. An initial 6 adult and 1 juvenile NSG mice were submitted for necropsy and histologic examination because of acute onset of diarrhea and death. The evaluation revealed typhlocolitis in 2 of the mice and tritrichomoniasis in all 7. Escherichia coli positive for polyketide synthase (pks+) and Kp were isolated from the intestines. Given a history of sepsis due to pks+ E. coli in NSG mice in our facilities and determination of its antimicrobial susceptibility, trimethoprim-sulfamethoxazole (TMP-SMX) was administered to the colony in the drinking water for 4 wk. After this intervention, an additional 21 mice became ill or died; 11 of these mice had suppurative pneumonia, meningoencephalitis, hepatitis, metritis, pyelonephritis, or sepsis. Kp was cultured from pulmonary abscesses or blood of 10 of the mice. Whole-genome sequencing (WGS) indicated that the Kp isolates contained genes associated with phenotypes found in pore-forming Kp isolates cultured from humans with ulcerative colitis and primary sclerosing cholangitis. None of the Kp isolates exhibited a hyperviscous phenotype, but 13 of 14 were resistant to TMP-SMX. Antimicrobial susceptibility testing indicated sensitivity of the Kp to enrofloxacin, which was administered in the drinking water. Antibiotic sensitivity profiles were confirmed by WGS of the Kp strains; key virulence and resistance genes to quaternary ammonia compounds were also identified. Enrofloxacin treatment resulted in a marked reduction in mortality, and the study using the NSG mice was completed successfully. Our findings implicate intestinal translocation of Kp as the cause of pneumonia and systemic infections in NSG mice and highlight the importance of identification of enteric microbial pathogens and targeted antibiotic selection when treating bacterial infections in immunocompromised mice.

Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Research Mouse Colonies.

Chlamydia muridarum (Cm) was detected in 2 colonies of mice with lymphoplasmacytic pulmonary infiltrates by using PCR and immunohistochemistry. This discovery was unexpected, as Cm infection had not been reported in laboratory mice since the 1940s. A Cm specific PCR assay was developed and testing implemented for the resident colonies of 8 vivaria from 3 academic institutions, 58 incoming mouse shipments from 39 academic institutions, and mice received from 55 commercial breeding colonies (4 vendors). To estimate Cm's global prevalence in research colonies, a database containing 11,387 metagenomic fecal microbiota samples from 120 institutions and a cohort of 900 diagnostic samples from 96 institutions were examined. Results indicate significant prevalence among academic institutions, with Cm detected in 63% of soiled bedding sentinels from 3 institutions; 33% of incoming mouse shipments from 39 academic institutions; 14% of 120 institutions submitting microbiota samples; and 16% of the diagnostic sample cohort. All samples from commercial breeding colonies were negative. In addition, naïve NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice exposed to Cm-shedding mice and/or their soiled bedding developed clinical disease at 21 to 28 d after exposure. These mice had a moderate-to-severe histiocytic and neutro- philic bronchointerstitial pneumonia, with their respiratory epithelium demonstrating inclusions, chlamydial major outer membrane protein immunostaining, and hybridization with a Cm reference sequence (GenBank accession no. U68436). Cm was isolated from lungs, cecum, and feces of a Cm-infected NSG mouse by using HeLa 229 cells. The considerable prevalence of Cm is likely due to widespread global interinstitutional distribution of unique mouse strains and failure to recognize that some of these mice were from enzootically infected colonies. Given that experimental Cm colonization of mice results in a robust immune response and, on occasion, pathology, natural infection may confound experimental results. Therefore, Cm should be excluded and eradicated from enzootically infected mouse colonies.