Publications by authors named "Carmen J Booth"

85 Publications

Ketogenic diet restrains aging-induced exacerbation of coronavirus infection in mice.

Elife 2021 Jun 21;10. Epub 2021 Jun 21.

Comparative Medicine and Immunobiology, Yale University School of Medicine, New Haven, United States.

Increasing age is the strongest predictor of risk of COVID-19 severity and mortality. Immunometabolic switch from glycolysis to ketolysis protects against inflammatory damage and influenza infection in adults. To investigate how age compromises defense against coronavirus infection, and whether a pro-longevity ketogenic-diet (KD) impacts immune-surveillance, we developed an aging model of natural murine beta coronavirus (mCoV) infection with mouse hepatitis virus strain-A59 (MHV-A59). When inoculated intranasally, mCoV is pneumotropic and recapitulates several clinical hallmarks of COVID-19 infection. Aged mCoV-A59-infected mice have increased mortality and higher systemic inflammation in the heart, adipose tissue and hypothalamus, including neutrophilia and loss of γδ T cells in lungs. Activation of ketogenesis in aged mice expands tissue protective γδ T cells, deactivates the NLRP3 inflammasome and decreases pathogenic monocytes in lungs of infected aged mice. These data establish harnessing of the ketogenic immunometabolic checkpoint as a potential treatment against coronavirus infection in the aged.
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http://dx.doi.org/10.7554/eLife.66522DOI Listing
June 2021

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency.

Mol Ther Methods Clin Dev 2020 Dec 22;19:486-495. Epub 2020 Oct 22.

Herman B Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA.

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized nucleotide sequences were employed. A comprehensive immunogenicity assessment using stimulation assays and various preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.
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http://dx.doi.org/10.1016/j.omtm.2020.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708868PMC
December 2020

Ketogenesis restrains aging-induced exacerbation of COVID in a mouse model.

bioRxiv 2020 Sep 12. Epub 2020 Sep 12.

Increasing age is the strongest predictor of risk of COVID-19 severity. Unregulated cytokine storm together with impaired immunometabolic response leads to highest mortality in elderly infected with SARS-CoV-2. To investigate how aging compromises defense against COVID-19, we developed a model of natural murine beta coronavirus (mCoV) infection with mouse hepatitis virus strain MHV-A59 (mCoV-A59) that recapitulated majority of clinical hallmarks of COVID-19. Aged mCoV-A59-infected mice have increased mortality and higher systemic inflammation in the heart, adipose tissue and hypothalamus, including neutrophilia and loss of γδ T cells in lungs. Ketogenic diet increases beta-hydroxybutyrate, expands tissue protective γδ T cells, deactivates the inflammasome and decreases pathogenic monocytes in lungs of infected aged mice. These data underscore the value of mCoV-A59 model to test mechanism and establishes harnessing of the ketogenic immunometabolic checkpoint as a potential treatment against COVID-19 in the elderly.

Highlights: - Natural MHV-A59 mouse coronavirus infection mimics COVID-19 in elderly.- Aged infected mice have systemic inflammation and inflammasome activation.- Murine beta coronavirus (mCoV) infection results in loss of pulmonary γδ T cells.- Ketones protect aged mice from infection by reducing inflammation.

Etoc Blurb: Elderly have the greatest risk of death from COVID-19. Here, Ryu et al report an aging mouse model of coronavirus infection that recapitulates clinical hallmarks of COVID-19 seen in elderly. The increased severity of infection in aged animals involved increased inflammasome activation and loss of γδ T cells that was corrected by ketogenic diet.
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http://dx.doi.org/10.1101/2020.09.11.294363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685240PMC
September 2020

A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis.

PLoS Pathog 2020 11 11;16(11):e1009030. Epub 2020 Nov 11.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America.

Lyme disease, the most common vector-borne illness in North America, is caused by the spirochete Borrelia burgdorferi. Infection begins in the skin following a tick bite and can spread to the hearts, joints, nervous system, and other organs. Diverse host responses influence the level of B. burgdorferi infection in mice and humans. Using a systems biology approach, we examined potential molecular interactions between human extracellular and secreted proteins and B. burgdorferi. A yeast display library expressing 1031 human extracellular proteins was probed against 36 isolates of B. burgdorferi sensu lato. We found that human Peptidoglycan Recognition Protein 1 (PGLYRP1) interacted with the vast majority of B. burgdorferi isolates. In subsequent experiments, we demonstrated that recombinant PGLYRP1 interacts with purified B. burgdorferi peptidoglycan and exhibits borreliacidal activity, suggesting that vertebrate hosts may use PGLYRP1 to identify B. burgdorferi. We examined B. burgdorferi infection in mice lacking PGLYRP1 and observed an increased spirochete burden in the heart and joints, along with splenomegaly. Mice lacking PGLYRP1 also showed signs of immune dysregulation, including lower serum IgG levels and higher levels of IFNγ, CXCL9, and CXCL10.Taken together, our findings suggest that PGLYRP1 plays a role in the host's response to B. burgdorferi and further demonstrate the utility of expansive yeast display screening in capturing biologically relevant interactions between spirochetes and their hosts.
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http://dx.doi.org/10.1371/journal.ppat.1009030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657531PMC
November 2020

Repeat tick exposure elicits distinct immune responses in guinea pigs and mice.

Ticks Tick Borne Dis 2020 11 2;11(6):101529. Epub 2020 Aug 2.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA; Howard Hughes Medical Institute. Chevy Chase, MD 20815, USA. Electronic address:

Ticks deposit salivary proteins into the skin during a bite to mediate acquisition of a blood meal. Acquired resistance to tick bites has been demonstrated to prevent Borrelia burgdorferi sensu lato (s.l.) transmission. However, the mechanism of resistance, as well as the protective antigens, have remained elusive. To address these unknowns, we utilized a guinea pig model of tick resistance and a mouse model of permissiveness. Guinea pigs developed immunity after multiple Ixodes scapularis tick infestations, characterized by rapid tick detachment and impaired feeding. In comparison, mice tolerated at least 6 infestations with no significant impact on feeding. We analyzed the bite sites by RNA-sequencing and histology, identifying several inflammatory pathways in tick immune animals, such as FcεRI signaling and complement activation, and activation of coagulation pathways that could impair local blood flow. Together, these results identify important pathways altered during tick rejection and potential tick proteins that could serve as vaccine candidates.
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http://dx.doi.org/10.1016/j.ttbdis.2020.101529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530331PMC
November 2020

An Ixodes scapularis Protein Disulfide Isomerase Contributes to Borrelia burgdorferi Colonization of the Vector.

Infect Immun 2020 11 16;88(12). Epub 2020 Nov 16.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.

causes Lyme disease, the most common tick-transmitted illness in North America. When feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, rotein isulfide somerase (IsPDIA3), enhances colonization of the tick gut. ticks in which has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on -infected mice. Moreover, administration of IsPDIA3 antiserum to -infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.
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http://dx.doi.org/10.1128/IAI.00426-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671890PMC
November 2020

High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss.

Elife 2020 04 9;9. Epub 2020 Apr 9.

Department of Physiology, Lee Gil Ya Cancer and Diabetes Institute, Gachon University College of Medicine, Incheon, Republic of Korea.

Phosphate overload contributes to mineral bone disorders that are associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and of negligible nutritional value in humans. However, the mechanism and adverse effects of high-phytate intake on Ca and phosphate absorption and homeostasis are unknown. Here, we show that excessive intake of phytate along with a low-Ca diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca diet and is a risk factor for phosphate overloading and for the development of crystal nephropathies and bone disease.
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http://dx.doi.org/10.7554/eLife.52709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145417PMC
April 2020

peptidoglycan is a persistent antigen in patients with Lyme arthritis.

Proc Natl Acad Sci U S A 2019 07 17;116(27):13498-13507. Epub 2019 Jun 17.

Microbial Sciences Institute, Yale University, West Haven, CT 06516;

Lyme disease is a multisystem disorder caused by the spirochete A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that has a chemically atypical PG (PG) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PG fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PG, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PG in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PG In addition, systemic administration of PG in BALB/c mice elicits acute arthritis. Altogether, our study identifies PG as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that sheds immunogenic PG fragments during growth suggests a potential role for PG in the immunopathogenesis of other Lyme disease manifestations.
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http://dx.doi.org/10.1073/pnas.1904170116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613144PMC
July 2019

Endothelial ERK1/2 signaling maintains integrity of the quiescent endothelium.

J Exp Med 2019 08 13;216(8):1874-1890. Epub 2019 Jun 13.

Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT

To define the role of ERK1/2 signaling in the quiescent endothelium, we induced endothelial knockout in adult mice. This resulted in a rapid onset of hypertension, a decrease in eNOS expression, and an increase in endothelin-1 plasma levels, with all mice dying within 5 wk. Immunostaining and endothelial fate mapping showed a robust increase in TGFβ signaling leading to widespread endothelial-to-mesenchymal transition (EndMT). Fibrosis affecting the cardiac conduction system was responsible for the universal lethality in these mice. Other findings included renal endotheliosis, loss of fenestrated endothelia in endocrine organs, and hemorrhages. An ensemble computational intelligence strategy, comprising deep learning and probabilistic programing of RNA-seq data, causally linked the loss of ERK1/2 in HUVECs in vitro to activation of TGFβ signaling, EndMT, suppression of eNOS, and induction of endothelin-1 expression. All in silico predictions were verified in vitro and in vivo. In summary, these data establish the key role played by ERK1/2 signaling in the maintenance of vascular normalcy.
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http://dx.doi.org/10.1084/jem.20182151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683988PMC
August 2019

Spontaneous Recurrent Absence Seizure-like Events in Wild-Caught Rats.

J Neurosci 2019 06 10;39(24):4829-4841. Epub 2019 Apr 10.

Department of Psychology and Neuroscience, University of Colorado, Boulder, Colorado 80309,

Absence epilepsy is a heritable human neurological disorder characterized by brief nonconvulsive seizures with behavioral arrest, moderate-to-severe loss of consciousness (absence), and distinct spike-wave discharges (SWDs) in the EEG and electrocorticogram (ECoG). Genetic models of this disorder have been created by selectively inbreeding rats for absence seizure-like events with similar electrical and behavioral characteristics. However, these events are also common in outbred laboratory rats, raising concerns about whether SWD/immobility accurately reflects absence epilepsy as opposed to "normal" rodent behavior. We hypothesized that, if SWD/immobility models absence seizures, it would not exist in wild-caught rats due to the pressures of natural selection. To test this hypothesis, we compared chronic video/electrocorticogram recordings from male and female wild-caught (Brown-Norway [BN]) rats to recordings from laboratory outbred BN, outbred Long-Evans, and inbred WAG/Rij rats (i.e., a model of absence epilepsy). Wild-caught BN rats displayed absence-like SWD/immobility events that were highly similar to outbred BN rats in terms of spike-wave morphology, frequency, diurnal rhythmicity, associated immobility, and sensitivity to the anti-absence drug, ethosuximide; however, SWD bursts were less frequent and of shorter duration in wild-caught and outbred BN rats than the outbred Long-Evans and inbred WAG/Rij strains. We conclude that SWD/immobility in rats does not represent absence seizures, although they appear to have many similarities. In wild rats, SWD/immobility appears to represent normal brain activity that does not reduce survival in natural environments, a conclusion that logically extends to outbred laboratory rats and possibly to those that have been inbred to model absence epilepsy. Spike-wave discharges (SWDs), behavioral arrest, and diminished consciousness are cardinal signs of seizures in human absence epilepsy and are used to model this disorder in inbred rats. These characteristics, however, are routinely found in outbred laboratory rats, leading to debate on whether SWD/immobility is a valid model of absence seizures. The SWD/immobility events in wild-caught rats appear equivalent to those found in outbred and inbred rat strains, except for lower incidence and shorter durations. Our results indicate that the electrophysiological and behavioral characteristics of events underlying hypothetical absence epilepsy in rodent models are found in wild rats captured in their natural environment. Other criteria beyond observation of SWDs and associated immobility are required to objectively establish absence epilepsy in rat models.
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http://dx.doi.org/10.1523/JNEUROSCI.1167-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561690PMC
June 2019

Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice.

Proc Natl Acad Sci U S A 2019 02 23;116(6):2200-2209. Epub 2019 Jan 23.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520;

Secondary hemophagocytic lymphohistiocytosis (sHLH) is a highly mortal complication associated with sepsis. In adults, it is often seen in the setting of infections, especially viral infections, but the mechanisms that underlie pathogenesis are unknown. sHLH is characterized by a hyperinflammatory state and the presence hemophagocytosis. We found that sequential challenging of mice with a nonlethal dose of viral toll-like receptor (TLR) agonist followed by a nonlethal dose of TLR4 agonist, but not other permutations, produced a highly lethal state that recapitulates many aspects of human HLH. We found that this hyperinflammatory response could be recapitulated in vitro in bone marrow-derived macrophages. RNA sequencing analyses revealed dramatic up-regulation of the red-pulp macrophage lineage-defining transcription factor SpiC and its associated transcriptional program, which was also present in bone marrow macrophages sorted from patients with sHLH. Transcriptional profiling also revealed a unique metabolic transcriptional profile in these macrophages, and immunometabolic phenotyping revealed impaired mitochondrial function and oxidative metabolism and a reliance on glycolytic metabolism. Subsequently, we show that therapeutic administration of the glycolysis inhibitor 2-deoxyglucose was sufficient to rescue animals from HLH. Together, these data identify a potential mechanism for the pathogenesis of sHLH and a potentially useful therapeutic strategy for its treatment.
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http://dx.doi.org/10.1073/pnas.1820704116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369774PMC
February 2019

Host-specific expression of Ixodes scapularis salivary genes.

Ticks Tick Borne Dis 2019 02 3;10(2):386-397. Epub 2018 Dec 3.

Section of Infectious Diseases, New Haven, CT, 06520, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, 20815, USA.

Ixodes scapularis vectors several pathogens including Borrelia burgdorferi, the agent of Lyme disease. Nymphal and larval stages, and the pathogens transmitted by I. scapularis are maintained in a zoonotic cycle involving rodent reservoir hosts, predominantly Peromyscus leucopus. Humans are not reservoir hosts, however, accidental encounters of infected ticks with humans, results in pathogen transmission to the human host. Laboratory models of non-reservoir hosts such as guinea pigs develop a strong immune response to tick salivary proteins and reject ticks upon repeated tick infestations. Anecdotal and scientific evidence suggests that humans that get frequent tick bites might also develop resistance to ticks. Mus musculus, the laboratory model of natural host, does not develop resistance to I. scapularis upon repeated tick infestations. Addressing this dichotomy in vector-host interaction, we present data that suggest that the salivary transcriptome and proteome composition is different in mouse and guinea pig-fed I. scapularis, and that these differences might contribute to differences in host immune responses. These findings reveal a new insight into vector-host interactions and offer a functional paradigm to better understand the phenomenon of acquired tick-resistance.
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http://dx.doi.org/10.1016/j.ttbdis.2018.12.001DOI Listing
February 2019

Glucose metabolism mediates disease tolerance in cerebral malaria.

Proc Natl Acad Sci U S A 2018 10 5;115(43):11042-11047. Epub 2018 Oct 5.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520;

Sickness behaviors are a conserved set of stereotypic responses to inflammatory diseases. We recently demonstrated that interfering with inflammation-induced anorexia led to metabolic changes that had profound effects on survival of acute inflammatory conditions. We found that different inflammatory states needed to be coordinated with corresponding metabolic programs to actuate tissue-protective mechanisms. Survival of viral inflammation required intact glucose utilization pathways, whereas survival of bacterial inflammation required alternative fuel substrates and ketogenic programs. We thus hypothesized that organismal metabolism would be important in other classes of infectious inflammation and sought to understand its role in the prototypic parasitic disease malaria. Utilizing the cerebral malaria model, ANKA (PbA) infection in C57BL/6J male mice, we unexpectedly found that inhibition of glycolysis using 2-deoxy glucose (2DG) conferred protection from cerebral malaria. Unlike vehicle-treated animals, 2DG-treated animals did not develop cerebral malaria and survived until ultimately succumbing to fatal anemia. We did not find any differences in parasitemia or pathogen load in affected tissues. There were no differences in the kinetics of anemia. We also did not detect differences in immune infiltration in the brain or in blood-brain barrier permeability. Rather, on pathological analyses performed on the entire brain, we found that 2DG prevented the formation of thrombi and thrombotic complications. Using thromboelastography (TEG), we found that 2DG-treated animals formed clots that were significantly less strong and stable. Together, these data suggest that glucose metabolism is involved in inflammation-induced hemostasis and provide a potential therapeutic target in treatment of cerebral malaria.
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http://dx.doi.org/10.1073/pnas.1806376115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205430PMC
October 2018

DNA damage response genes mark the early transition from colitis to neoplasia in colitis-associated colon cancer.

Gene 2018 Nov 16;677:299-307. Epub 2018 Aug 16.

Department of Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA. Electronic address:

Chronic intestinal inflammation predisposes patients with Inflammatory Bowel Disease (IBD) to Colitis-Associated Cancer (CAC). In the setting of chronic inflammation, microsatellite instability (MSI) results from early loss of DNA damage response (DDR) genes, ultimately leading to tumor formation. Despite continued efforts to improve early detection of high risk, pre-dysplastic regions in IBD patients, current macroscopic and genetic surveillance modalities remain limited. Therefore, understanding the regulation of key DDR genes in the progression from colitis to cancer may improve molecular surveillance of CAC. To evaluate DDR gene regulation in the transition from colitis to tumorigenesis, we utilized the well-established Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) pre-clinical murine model of CAC in C57BL/6 mice. In order to assess colonic tumor burden in the setting of mutagen and intestinal irritation, tumors were visualized and graded in real time through high-resolution murine colonoscopy. Upon sacrifice, colons were opened and assessed for macroscopic tumor via high magnification surgical lenses (HMSL). Tissues were then sectioned and separated into groups based on the presence or absence of macroscopically visible tumor. Critical DDR genes were evaluated by semi-quantitative RT-PCR. Interestingly, colon tissue with macroscopically visible tumor (MVT) and colon tissue prior to observable tumor (the non-macroscopically visible tumor-developing group, NMVT) were identical in reduced mRNA expression of mlh1, anapc1, and ercc4 relative to colitic mice without mutagen, or those receiving mutagen alone. Colitis alone was sufficient to reduce colonic ercc4 expression when compared to NMVT mice. Therefore, reduced ercc4 expression may mark the early transition to CAC in a pre-clinical model, with expression reduced prior to the onset of observable tumor. Moreover, the expression of select DDR genes inversely correlated with chronicity of inflammatory disease. These data suggest ercc4 expression may define early stages in the progression to CAC.
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http://dx.doi.org/10.1016/j.gene.2018.08.016DOI Listing
November 2018

In Vivo Reactive Oxygen Species Detection With a Novel Positron Emission Tomography Tracer, F-DHMT, Allows for Early Detection of Anthracycline-Induced Cardiotoxicity in Rodents.

JACC Basic Transl Sci 2018 Jun 30;3(3):378-390. Epub 2018 May 30.

Section of Cardiovascular Medicine, Department of Medicine, Yale Translational Research Imaging Center, Yale School of Medicine, New Haven, Connecticut.

Reactive oxygen species (ROS) are involved in doxorubicin-induced cardiotoxicity. The authors investigated the efficacy of F-DHMT, a marker of ROS, for early detection of doxorubicin-induced cardiotoxicity in rats. Echocardiography was performed at baseline and 4, 6, and 8 weeks post-doxorubicin initiation, whereas in vivo superoxide production was measured at 4 and 6 weeks with F-DHMT positron emission tomography. Left ventricular ejection fraction (LVEF) was not significantly decreased until 6 weeks post-doxorubicin treatment, whereas myocardial superoxide production was significantly elevated at 4 weeks. F-DHMT imaging detected an elevation in cardiac superoxide production before a fall in LVEF in rodents and may allow for early cardiotoxicity detection in cancer patients.
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http://dx.doi.org/10.1016/j.jacbts.2018.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058999PMC
June 2018

Lack of Effect of Murine Astrovirus Infection on Dextran Sulfate-induced Colitis in NLRP3-deficient Mice.

Comp Med 2017 Oct;67(5):400-406

Section of Comparative Medicine, Animal Resources Center, Yale University, New Haven, Connecticut.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. MuAstV is found predominantly in the gastrointestinal tract of mice. Human and turkey astroviruses have been shown to disrupt tight junctions in the intestinal epithelium. The potential of MuAstV to alter research results was tested in a well-established dextran sodium sulfate (DSS)-induced colitis model in Nod-like receptor 3 (NLRP3)-deficient mice. This model offers a direct approach to determine whether MuAstV, as a component of the mouse microbiome, contributes to the issue of poor reproducibility in murine inflammatory bowel disease research. In this model, defective inflammasome activation causes loss of epithelial integrity, resulting in leakage of intestinal bacteria and colitis. Our goal was to determine whether MuAstV, which also may affect intestinal permeability, altered the onset or severity of colitis. Male and female mice (age, 8 to 12 wk) homozygous or heterozygous for an NLRP3 mutation were inoculated orally with MuAstV or mock-inoculated with media 3 or 20 d prior to being exposed to 2% DSS in their drinking water for 9 d. MuAstV infection alone did not cause clinical signs or histopathologic changes in NLRP3-/- or NLRP3+/- mice. No significant difference was seen in weight loss, clinical disease, intestinal inflammation, edema, hyperplasia, or mucosal ulceration between MuAstV- infected and mock-infected mice that received 2% DSS for 9 d. Therefore, MuAstV does not appear to be a confounding variable in the DSS colitis model in NLRP3 mice.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621567PMC
October 2017

Murine Astrovirus Infection and Transmission in Neonatal CD1 Mice.

J Am Assoc Lab Anim Sci 2017 Jul;56(4):402-411

Section of Comparative Medicine, School of Medicine, Animal Resources Center, Yale University.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. Our goal was to determine the duration of MuAstV infection, susceptibility of pups, and efficacy of soiled-bedding sentinels and environmental monitoring. Eight CD1 dams and their litters of 3-d-old pups and 8 CD1 dams and their litters of 13-d-old mice were inoculated orally with MuAstV. Neither dams nor offspring demonstrated any clinical signs, and MuAstV had little to no effect on weight gain in pups. MuAstV RNA was detected in feces from 15 of the 16 dams through postnatal day (PND) 21, and 9 dams were still shedding MuAstV at PND 42. MuAstV RNA was highest in intestines of mice. Low levels of MuAstV RNA were sporadically detected in the spleen, liver, and kidney. MuAstV was detected in 97% of feces from 3- to 9-wk-old mice born to infected dams. Several weanlings became pregnant, and intestines from their pups were MuAstV-negative at PND 0 through 5. Weekly swabs of cages housing MuAstV-infected mice were MuAstV-positive through PND 42. Swabs of the rear exhaust manifold of the ventilated rack were MuAstV-positive at 21 through 56 d after inoculation. In addition, 98% of sentinels that received soiled bedding from dams and their litters and 83% of sentinels that received soiled bedding from weaned mice were MuAstV-positive. Feces from most sentinels exposed to soiled bedding that had been stored for 1, 2 or 3 wk before addition of the sentinels were MuAstV-positive.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517330PMC
July 2017

Voluntary Control of Epileptiform Spike-Wave Discharges in Awake Rats.

J Neurosci 2017 06 18;37(24):5861-5869. Epub 2017 May 18.

Department of Psychology and Neuroscience, University of Colorado, Boulder, Colorado 80309,

Genetically inherited absence epilepsy in humans is typically characterized by brief (seconds) spontaneous seizures, which involve spike-wave discharges (SWDs) in the EEG and interruption of consciousness and ongoing behavior. Genetic (inbred) models of this disorder in rats have been used to examine mechanisms, comorbidities, and antiabsence drugs. SWDs have also been proposed as models of complex partial seizures (CPSs) following traumatic brain injury (post-traumatic epilepsy). However, the ictal characteristics of these rat models, including SWDs and associated immobility, are also prevalent in healthy outbred laboratory rats. We therefore hypothesized that SWDs are not always associated with classically defined absence seizures or CPSs. To test this hypothesis, we used operant conditioning in male rats to determine whether outbred strains, Sprague Dawley and Long-Evans, and/or the inbred strain (a rat model of heritable human absence epilepsy) could exercise voluntary control over these epileptiform events. We discovered that both inbred and outbred rats could shorten the duration of SWDs to obtain a reward. These results indicate that SWD and associated immobility in rats may not reflect the obvious cognitive/behavioral interruption classically associated with absence seizures or CPSs in humans. One interpretation of these results is that human absence seizures and perhaps CPSs could permit a far greater degree of cognitive capacity than often assumed and might be brought under voluntary control in some cases. However, these results also suggest that SWDs and associated immobility may be nonepileptic in healthy outbred rats and reflect instead voluntary rodent behavior unrelated to genetic manipulation or to brain trauma. Our evidence that inbred and outbred rats learn to control the duration of spike-wave discharges (SWDs) suggests a voluntary behavior with maintenance of consciousness. If SWDs model mild absence seizures and/or complex partial seizures in humans, then an opportunity may exist for operant control complementing or in some cases replacing medication. Their equal occurrence in outbred rats also implies a major potential confound for behavioral neuroscience experiments, at least in adult rats where SWDs are prevalent. Alternatively, the presence and voluntary control of SWDs in healthy outbred rats could indicate that these phenomena do not always model heritable absence epilepsy or post-traumatic epilepsy in humans, and may instead reflect typical rodent behavior.
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http://dx.doi.org/10.1523/JNEUROSCI.3235-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596506PMC
June 2017

Loss of Dynamin 2 GTPase function results in microcytic anaemia.

Br J Haematol 2017 08 3;178(4):616-628. Epub 2017 May 3.

Australian Centre for Blood Diseases, Central Clinical School, Monash University and Alfred Health, Melbourne, Vic., Australia.

In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2 cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2 mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.
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http://dx.doi.org/10.1111/bjh.14709DOI Listing
August 2017

Quantification of Tumor Hypoxic Fractions Using Positron Emission Tomography with [F]Fluoromisonidazole ([F]FMISO) Kinetic Analysis and Invasive Oxygen Measurements.

Mol Imaging Biol 2017 12;19(6):893-902

Department of Therapeutic Radiology, Yale University School of Medicine, P.O. Box 208040, New Haven, CT, 06520-8040, USA.

Purpose: The purpose of this study is to use dynamic [F]fluoromisonidazole ([F]FMISO) positron emission tomography (PET) to compare estimates of tumor hypoxic fractions (HFs) derived by tracer kinetic modeling, tissue-to-blood ratios (TBR), and independent oxygen (pO) measurements.

Procedures: BALB/c mice with EMT6 subcutaneous tumors were selected for PET imaging and invasive pO measurements. Data from 120-min dynamic [F]FMISO scans were fit to two-compartment irreversible three rate constant (K , k , k ) and Patlak models (K ). Tumor HFs were calculated and compared using K , k , TBR, and pO values. The clinical impact of each method was evaluated on [F]FMISO scans for three non-small cell lung cancer (NSCLC) radiotherapy patients.

Results: HFs defined by TBR (≥1.2, ≥1.3, and ≥1.4) ranged from 2 to 85 % of absolute tumor volume. HFs defined by K (>0.004 ml min cm) and k (>0.008 min) varied from 9 to 85 %. HF quantification was highly dependent on metric (TBR, k , or K ) and threshold. HFs quantified on human [F]FMISO scans varied from 38 to 67, 0 to 14, and 0.1 to 27 %, for each patient, respectively, using TBR, k , and K metrics.

Conclusions: [F]FMISO PET imaging metric choice and threshold impacts hypoxia quantification reliability. Our results suggest that tracer kinetic modeling has the potential to improve hypoxia quantification clinically as it may provide a stronger correlation with direct pO measurements.
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http://dx.doi.org/10.1007/s11307-017-1083-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640490PMC
December 2017

A pathogenic role for T cell-derived IL-22BP in inflammatory bowel disease.

Science 2016 10;354(6310):358-362

I. Medizinische Klinik und Poliklinik, Universitätsklinikum Hamburg-Eppendorf, 20246 Hamburg, Germany.

Intestinal inflammation can impair mucosal healing, thereby establishing a vicious cycle leading to chronic inflammatory bowel disease (IBD). However, the signaling networks driving chronic inflammation remain unclear. Here we report that CD4 T cells isolated from patients with IBD produce high levels of interleukin-22 binding protein (IL-22BP), the endogenous inhibitor of the tissue-protective cytokine IL-22. Using mouse models, we demonstrate that IBD development requires T cell-derived IL-22BP. Lastly, intestinal CD4 T cells isolated from IBD patients responsive to treatment with antibodies against tumor necrosis factor-α (anti-TNF-α), the most effective known IBD therapy, exhibited reduced amounts of IL-22BP expression but still expressed IL-22. Our findings suggest that anti-TNF-α therapy may act at least in part by suppressing IL-22BP and point toward a more specific potential therapy for IBD.
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http://dx.doi.org/10.1126/science.aah5903DOI Listing
October 2016

Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation.

Cell 2016 Sep;166(6):1512-1525.e12

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

Acute infections are associated with a set of stereotypic behavioral responses, including anorexia, lethargy, and social withdrawal. Although these so-called sickness behaviors are the most common and familiar symptoms of infections, their roles in host defense are largely unknown. Here, we investigated the role of anorexia in models of bacterial and viral infections. We found that anorexia was protective while nutritional supplementation was detrimental in bacterial sepsis. Furthermore, glucose was necessary and sufficient for these effects. In contrast, nutritional supplementation protected against mortality from influenza infection and viral sepsis, whereas blocking glucose utilization was lethal. In both bacterial and viral models, these effects were largely independent of pathogen load and magnitude of inflammation. Instead, we identify opposing metabolic requirements tied to cellular stress adaptations critical for tolerance of differential inflammatory states. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2016.07.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555589PMC
September 2016

miR-34a Silences c-SRC to Attenuate Tumor Growth in Triple-Negative Breast Cancer.

Cancer Res 2016 Feb 16;76(4):927-39. Epub 2015 Dec 16.

Institute for RNA Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell-like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755913PMC
February 2016

AID-associated DNA repair pathways regulate malignant transformation in a murine model of BCL6-driven diffuse large B-cell lymphoma.

Blood 2016 Jan 18;127(1):102-12. Epub 2015 Sep 18.

Yale Cancer Center, Section of Hematology.

Somatic hypermutation and class-switch recombination of the immunoglobulin (Ig) genes occur in germinal center (GC) B cells and are initiated through deamination of cytidine to uracil by activation-induced cytidine deaminase (AID). Resulting uracil-guanine mismatches are processed by uracil DNA glycosylase (UNG)-mediated base-excision repair and MSH2-mediated mismatch repair (MMR) to yield mutations and DNA strand lesions. Although off-target AID activity also contributes to oncogenic point mutations and chromosome translocations associated with GC and post-GC B-cell lymphomas, the role of downstream AID-associated DNA repair pathways in the pathogenesis of lymphoma is unknown. Here, we show that simultaneous deficiency of UNG and MSH2 or MSH2 alone causes genomic instability and a shorter latency to the development of BCL6-driven diffuse large B-cell lymphoma (DLBCL) in a murine model. The additional development of several BCL6-independent malignancies in these mice underscores the critical role of MMR in maintaining general genomic stability. In contrast, absence of UNG alone is highly protective and prevents the development of BCL6-driven DLBCL. We further demonstrate that clonal and nonclonal mutations arise within non-Ig AID target genes in the combined absence of UNG and MSH2 and that DNA strand lesions arise in an UNG-dependent manner but are offset by MSH2. These findings lend insight into a complex interplay whereby potentially deleterious UNG activity and general genomic instability are opposed by the protective influence of MSH2, producing a net protective effect that promotes immune diversification while simultaneously attenuating malignant transformation of GC B cells.
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http://dx.doi.org/10.1182/blood-2015-02-628164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705602PMC
January 2016

Haptoglobin enhances cardiac transplant rejection.

Circ Res 2015 May 23;116(10):1670-9. Epub 2015 Mar 23.

From the Department of Internal Medicine (H.S., E.H., D.N.M., C.K.W., D.R.G.), Department of Immunobiology (H.S., D.N.M., C.K.W., D.R.G.), W.M. Keck Biotechnology Resource Laboratory (C.M.C., L.M.C.), Center for Medical Informatics (C.B.), and Section of Comparative Medicine (I.B.S., C.J.B.), Yale School of Medicine, New Haven, CT; Sciomix, Woodbridge, CT (C.B.); Department of Surgery (D.K.) and Department of Immunology (D.K.), Washington University School of Medicine, St Louis, MO.

Rationale: Early graft inflammation enhances both acute and chronic rejection of heart transplants, but it is unclear how this inflammation is initiated.

Objective: To identify specific inflammatory modulators and determine their underlying molecular mechanisms after cardiac transplantation.

Methods And Results: We used a murine heterotopic cardiac transplant model to identify inflammatory modulators of early graft inflammation. Unbiased mass spectrometric analysis of cardiac tissue before and ≤72 hours after transplantation revealed that 22 proteins including haptoglobin, a known antioxidant, are significantly upregulated in our grafts. Through the use of haptoglobin-deficient mice, we show that 80% of haptoglobin-deficient recipients treated with perioperative administration of the costimulatory blocking agent CTLA4 immunoglobulin exhibited >100-day survival of full major histocompatibility complex mismatched allografts, whereas all similarly treated wild-type recipients rejected their transplants by 21 days after transplantation. We found that haptoglobin modifies the intra-allograft inflammatory milieu by enhancing levels of the inflammatory cytokine interleukin-6 and the chemokine MIP-2 (macrophage inflammatory protein 2) but impair levels of the immunosuppressive cytokine interleukin-10. Haptoglobin also enhances dendritic cell graft recruitment and augments antidonor T-cell responses. Moreover, we confirmed that the protein is present in human cardiac allograft specimens undergoing acute graft rejection.

Conclusions: Our findings provide new insights into the mechanisms of inflammation after cardiac transplantation and suggest that, in contrast to its prior reported antioxidant function in vascular inflammation, haptoglobin is an enhancer of inflammation after cardiac transplantation. Haptoglobin may also be a key component in other sterile inflammatory conditions.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.305406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426092PMC
May 2015

Frostbite protection in mice expressing an antifreeze glycoprotein.

PLoS One 2015 25;10(2):e0116562. Epub 2015 Feb 25.

Department of Internal Medicine, Section of Infectious Diseases, Yale University School of Medicine, New Haven, CT, United States of America; Howard Hughes Medical Institute, Chevy Chase, MD, United States of America.

Ectotherms in northern latitudes are seasonally exposed to cold temperatures. To improve survival under cold stress, they use diverse mechanisms to increase temperature resistance and prevent tissue damage. The accumulation of anti-freeze proteins that improve cold hardiness occurs in diverse species including plants, arthropods, fish, and amphibians. We previously identified an Ixodes scapularis anti-freeze glycoprotein, named IAFGP, and demonstrated its cold protective function in the natural tick host and in a transgenic Drosophila model. Here we show, in a transgenic mouse model expressing an anti-freeze glycoprotein, that IAFGP protects mammalian cells and mice from cold shock and frostbite respectively. Transgenic skin samples showed reduced cell death upon cold storage ex vivo and transgenic mice demonstrated increased resistance to frostbite injury in vivo. IAFGP actively protects mammalian tissue from freezing, suggesting its application for the prevention of frostbite, and other diseases associated with cold exposure.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116562PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340617PMC
January 2016

Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis.

Breast Cancer Res 2014 Dec 17;16(6):501. Epub 2014 Dec 17.

Department of Pathology, Yale School of Medicine, P.O.Box 208023, New Haven, CT, 06520, USA.

Introduction: Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer.

Methods: We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed.

Results: Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform.

Conclusions: Together, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.
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http://dx.doi.org/10.1186/s13058-014-0501-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303208PMC
December 2014

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

Hum Gene Ther 2015 Feb 21;26(2):69-81. Epub 2015 Jan 21.

1 Gene Therapy Center, University of North Carolina , Chapel Hill, NC 27599.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100-500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).
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http://dx.doi.org/10.1089/hum.2014.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326268PMC
February 2015

Evaluation of developmental toxicity of propylthiouracil and methimazole.

Birth Defects Res B Dev Reprod Toxicol 2014 Aug 30;101(4):300-7. Epub 2014 Jun 30.

Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida.

Background: Propylthiouracil (PTU) and methimazole (MMI) are antithyroid drugs used to treat hyperthyroidism. Despite the widespread use of PTU and MMI during pregnancy, modest clinical data and less animal data are available on the teratogenic potential of these drugs.

Methods: We evaluated the teratogenicity of in utero exposure to PTU or MMI in mice and rats. First, pregnant C57Bl/6 mice were treated daily with PTU (10 or 100 mg/kg), MMI (2 or 20 mg/kg), or vehicle from gestation day (GD) 6 to 16. GD 18 fetuses were evaluated for gross and histopathological abnormalities. Next, pregnant Sprague-Dawley rats were treated daily with PTU (50 or 100 mg/kg), MMI (10 or 20 mg/kg), or vehicle from GD 6 to 19, followed by evaluation for gross and histopathological abnormalities at GD 20.

Results: In mice treated with PTU or MMI, no significant histopathological abnormalities or external gross malformations, and no adverse effects on placental weight, litter size, resorption rates, or fetal weight were observed at GD 18. In rats, no adverse effects on litter size, placental weights, or maternal body weights were observed with either PTU or MMI treatment. PTU treatment (50 and 100 mg/kg) and MMI (10 mg/kg) treatment resulted in a decrease in crown-rump length in rat fetuses but no external gross malformations or histopathological abnormalities were observed.

Conclusion: We did not observe either gross external malformations or histopathological malformations in mice or rats treated long-term with high doses of PTU or MMI during pregnancy.
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http://dx.doi.org/10.1002/bdrb.21113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295064PMC
August 2014

Dual role for Fcγ receptors in host defense and disease in Borrelia burgdorferi-infected mice.

Front Cell Infect Microbiol 2014 11;4:75. Epub 2014 Jun 11.

Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine New Haven, CT, USA.

Arthritis in mice infected with the Lyme disease spirochete, Borrelia burgdorferi, results from the influx of innate immune cells responding to the pathogen in the joint and is influenced in part by mouse genetics. Production of inflammatory cytokines by innate immune cells in vitro is largely mediated by Toll-like receptor (TLR) interaction with Borrelia lipoproteins, yet surprisingly mice deficient in TLR2 or the TLR signaling molecule MyD88 still develop arthritis comparable to that seen in wild type mice after B. burgdorferi infection. These findings suggest that other, MyD88-independent inflammatory pathways can contribute to arthritis expression. Clearance of B. burgdorferi is dependent on the production of specific antibody and phagocytosis of the organism. As Fc receptors (FcγR) are important for IgG-mediated clearance of immune complexes and opsonized particles by phagocytes, we examined the role that FcγR play in host defense and disease in B. burgdorferi-infected mice. B. burgdorferi-infected mice deficient in the Fc receptor common gamma chain (FcεRγ(-/-) mice) harbored ~10 fold more spirochetes than similarly infected wild type mice, and this was associated with a transient increase in arthritis severity. While the elevated pathogen burdens seen in B. burgdorferi-infected MyD88(-/-) mice were not affected by concomitant deficiency in FcγR, arthritis was reduced in FcεRγ(-/-) MyD88(-/-) mice in comparison to wild type or single knockout mice. Gene expression analysis from infected joints demonstrated that absence of both MyD88 and FcγR lowers mRNA levels of proteins involved in inflammation, including Cxcl1 (KC), Xcr1 (Gpr5), IL-1beta, and C reactive protein. Taken together, our results demonstrate a role for FcγR-mediated immunity in limiting pathogen burden and arthritis in mice during the acute phase of B. burgdorferi infection, and further suggest that this pathway contributes to the arthritis that develops in B. burgdorferi-infected MyD88(-/-) mice.
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http://dx.doi.org/10.3389/fcimb.2014.00075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052197PMC
September 2014