Genital infection of rats with Mycoplasma pulmonis causes adverse pregnancy outcome and can result in in utero spread of infection to the fetus. The current study was designed to determine whether the stage of pregnancy when infection occurs influences pregnancy outcome. Rats were inoculated with 3 ؋ 10 7 CFU of M. pulmonis at 10 days prior to breeding (؊10) or at gestational day (gd) 11 or 14 and were necropsied at gd 11, 14, or 18 or within 24 h of parturition (term). Control rats received sterile broth. M. pulmonis was isolated from the placenta, amniotic fluid, or fetal tissues only from rats infected prior to breeding (P < 0.001). All infected rats had significantly more loss of pups than did control rats (P < 0.006), but rats infected prior to breeding or at the beginning of the third trimester (gd 14) were much more likely to have fetal losses. Rats infected in the early second trimester after implantation (gd 11) did not experience severe losses. Litter sizes, total litter weight, and individual pup weight from all infected rats, regardless of gestational stage when infected, were significantly smaller than those of control rats (P < 0.001). On the basis of the results of this study, we conclude that the time of infection plays a major role in determination of pregnancy outcome and spread of infection from the genital tract to the respiratory tract. Mycoplasma pulmonis is a common pathogen of mice and rats in many conventionally maintained colonies (3-5, 14). In addition to its role in respiratory disease, M. pulmonis is responsible for genital infections and infertility (5, 7, 17, 18). It has been estimated that M. pulmonis infection decreases rat birth rate 50 to 100% (5, 17). We have demonstrated the adverse impact of genital infection on pregnancy outcome in experimentally infected Sprague-Dawley (SD) rats (17). Genital infection prior to breeding resulted in increased fetal resorptions and an increased number of dams with no liveborn pups (17). Furthermore, individual pup weight, litter size, and litter weight were also decreased in infected rats. In a second study, we demonstrated that M. pulmonis could invade the placenta, breach the placental barrier, and establish an amniotic fluid infection by gestational day (gd) 14 (18). M. pulmonis was isolated from the oropharynx as well as lungs of fetuses at gd 18, confirming in utero transmission (18). Histological evidence was compatible with an active infection characterized by placentitis, amnionitis, and occasional mild fetal bronchopneumonia (18). M. pulmonis is an ideal candidate for a model of intrauterine infection. First, it is a naturally occurring disease. Second, the infection can be established by intravaginal inoculation, without requiring extensive manipulation of the animal. Third, the natural course of disease in the rat is similar to that predicted for human pathogens, i.e., an ascending infection that breaches the placental barrier and establishes as an amnionitis. Finally, a strong database exists for normal reproductive physiology and...

Specific-pathogen-free (SPF) female Sprague-Dawley rats were infected by intravaginal inoculation with 3 x 107 CFU of Mycoplasma pulnonis X1048 in 0.1 ml of Frey's broth or with an equal volume of sterile Frey's broth. A minimum of 10 days postinfection, rats were bred to noninfected males. Rats were necropsied at days 11, 14, and 18 of gestation and within 24 h of parturition. Throughout pregnancy, at least 50%Xo of rats remained infected in the lower genital tract. At parturition, the major site of colonization was the respiratory tract (P = 0.02). M. pulmonis was not isolated from any site of any control rat. Pregnancy outcome was adversely affected by infection with M. pulmonis. Infected rats had significantly smaller litter sizes at day 18 of gestation (P c 0.01) and at term (P c 0.004). No statistically significant differences among the gestational stages in infected rats were noted for litter size. Total litter weight is a reflection of individual pup weight and of the number of pups born. Therefore, it was obvious that infected rats would have a significantly lower (P _ 0.008) total litter weight than noninfected controls. However, when individual pup weights were considered, infected pups (n = 49) also had significantly lower (P 5 0.0001) birth weights than did noninfected controls (n = 68). The incidence of an adverse pregnancy outcome at term (stillbirths, macerated fetuses, or resorptions) was higher (P 5 0.01) in infected rats than in noninfected control rats. No stillborn pups or macerated fetuses were observed in any control term rats (n = 5). All control rats had live-born pups. Three infected rats had no live-born offspring. Resorptions were more common in infected rats than in control rats (P 5 0.01). The mean number of resorptions per rat was greater in rats which went to term than in rats necropsied during gestation, indicating that the severity of disease was progressive. The rat is frequently the laboratory animal of choice for a wide variety of reproductive studies, and the experimental parameters that are most often measured (litter size, pup weight, and neonatal survival) were all adversely affected by genital mycoplasmosis. Genital mycoplasmosis is important as an animal model for the interaction of infectious agents and the host during pregnancy as well as in its own right as a confounding variable affecting research projects which use the rat as a model to study reproductive function and physiology. * Corresponding author. t Journal series article R-02836 of the Florida Agricultural Experiment Station.dacryoadenitis virus, reovirus type 3, Kilham rat virus, Hantaan virus, M. pulmonis, respiratory and enteric bacterial pathogens, endoparasites, and ectoparasites. The rats were shipped in filter containers to ensure their specificpathogen-free status.Husbandry. All rats were housed in Microisolator (Lab Products, Inc., Maywood, N.J.) cages to maintain the various infection groups in separate isolation. Rats received 633 on August 1, 2020 by guest

Genital mycoplasmosis is important as an animal model for the interaction between infectious agents and the host during pregnancy as well as in its own right as a confounding variable affecting research projects in which the rat is used as a model to study reproductive function and physiology. We report the in utero transmission of Mycoplasma pulmonis and the development of placentitis, amnionitis, and mild fetal bronchopneumonia in Sprague-Dawley rats. A minimum of 10 days prior to breeding, specific-pathogen-free female Sprague-Dawley rats were infected by intravaginal inoculation with 3 x 107 CFU ofM. pulmonis X1048 or with an equal volume of sterile broth. Rats and fetuses were subjected to necropsy at days 11, 14, and 18 of gestation. M. pulmonis was able to invade the placenta, cross the placental barrier, and establish an amniotic fluid infection by gestational day 14. It was isolated from the oropharynx and lungs of fetuses at gestational day 18. The placenta was more frequently colonized than amniotic fluid, followed by the fetal oropharynx and lungs, supporting an ascending route of infection. Histopathological evidence also support an active infection, with lesions compatible with placentitis, amnionitis, and mild fetal bronchopneumonia. M. pulmonis can traverse the placenta, resulting in infection of the amniotic fluid and in utero transmission of the microorganism to the developing fetus.

The COVID-19 pandemic has drastically impacted work, economy, and way of life. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into pre-existing immunity, virus transmission dynamics, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient reliable detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. Very low optical densities from samples added to buffer coated wells at as low as a 1:5 dilution are reported using this ‘BU ELISA’ method. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA) in 44 and 100 percent of pre-pandemic subjects, respectively, and the magnitude of these antibodies tracked with antibody levels of analogous viral proteins from endemic coronavirus (eCoV) strains. The disease status (HIV, SLE) of unexposed subjects was not linked with SARS-CoV-2 reactive antibody levels; however, quantities were significantly lower in subjects over 70 years of age compared with younger counterparts. Also, we measured SARS-CoV-2 RBD- and N- specific IgM, IgG, and IgA antibodies from 29 SARS-CoV-2 infected individuals at varying disease states, including 10 acute COVID-19 hospitalized subjects with negative serology results by the EUA approved Abbott IgG chemiluminescent microparticle immunoassay. Measurements of SARS-CoV-2 RBD- and N- specific IgM, IgG, IgA levels measured by the BU ELISA revealed higher signal from 9 of the 10 Abbott test negative COVID-19 subjects than all pre-pandemic samples for at least one antibody specificity/isotype, implicating improved serologic identification of SARS-CoV-2 infection via multi-parameter, high sensitive antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and immunity and has promising implications for improved detection of all analytes measurable by this platform.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-β, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-β. Truncating the intracellular signaling domain from TGF-β receptor (TGFβR) II produces a dominant-negative receptor (dnTGFβRII) that dimerizes with endogenous TGFβRI to form a receptor that can bind TGF-β but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02–restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157–165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254–262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-β inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFβRII (e.g., GSK3845097). TGF-β isoforms and a panel of TGF-β–associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-β–positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non–small cell lung cancer setting. Coexpression of dnTGFβRII may therefore improve the efficacy of TCR-transduced T cells.

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The COVID-19 pandemic has significantly impacted work, economy, and way of life. The SARS-CoV-2 virus displays unique features including widely varying symptoms and outcomes between infected individuals. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into virus transmission dynamics, pre-existing cross-reactive immunity, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. This BU ELISA method exhibits very low signal from plasma or serum samples added to uncoated wells at as low as a 1:5 dilution. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (NP) reactive antibodies from blood samples drawn prior to May 2019. Of our pre-pandemic cohort, no SARS-CoV-2 RBD-reactive IgG antibodies were detected in subjects over 70 years of age, and SARS-CoV-2 NP-reactive antibodies were present at similar levels to infected subjects in some individuals and very low in others. Also, samples drawn in May 2020 from two individuals with no symptoms or no known virus exposure contained SARS-CoV-2 RBD-reactive antibodies at intermediate amounts compared with other subject groups (higher than pre-pandemic and lower than confirmed SARS-CoV-2 infected). The one asymptomatic SARS-CoV-2 convalescent subject in our study possessed comparable amounts of SARS-CoV-2 NP-specific IgM and IgG but drastically lower IgA than the symptomatic counterparts. Also, our assay detected positive signal from samples that gave negative results in a commercially available Lateral Flow Device (LFD) and the EUA approved Abbott IgG chemiluminescent microparticle immunoassay for SARS-CoV-2 antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and has promising implications for improved detection of all analytes measurable by this platform.

Microscopic vascular invasion (VI) is predictive of recurrence in stage I lung adenocarcinoma (LUAD) but is difficult to assess in resection specimens and cannot be accurately predicted prior to surgery. Thus, new biomarkers are needed to identify this aggressive subset of stage I LUAD tumors. To assess molecular and tumor microenvironment (TME) features associated with angioinvasive LUAD we profiled 171 resected stage I tumors with and without VI by RNA-seq, including 16 tumors by high-resolution spatial transcriptomics (10x Genomics Visium). Visium capture areas were selected by an experienced thoracic pathologist to include invasive foci, tumor regions distal to foci, and tumors without invasive foci. We identified a robust molecular signature of VI from the RNA-seq data containing subclusters of genes involved in hallmark programs of tumor suppression, EMT, angiogenesis, growth and metabolism. This VI-associated signature increases across the spectrum of indolent to aggressive stage I LUAD histopathology and is predictive of recurrence-free survival. Analysis of 43,421 Visium spots across 16 tumors revealed high inter-tumor patient heterogeneity, with most spots clustering by tumor identity, suggesting tumor-intrinsic properties. Scoring the Visium data for VI signature expression revealed spatial variability around VI foci, with distinct spatial distributions of different signature subclusters among tumor and stromal compartments within and adjacent to regions of tumor intravasation. We found increased expression of the bulk VI signature in invasive tumors, regardless of whether the capture area contained the invasive focus. We independently verified this finding by performing laser-capture microdissection and RNA-seq from invaded vessel regions and uninvaded parenchyma of 8 additional tumors with VI and tumor regions of non-VI tumors. Given increased signature expression in regions of invasive tumors at a distance from VI foci, we leveraged our bulk RNA-seq dataset to develop a transcriptomic predictor of VI. We applied a nested cross-validation approach within a training cohort to select a machine learning model. We then evaluated the performance of our model in an independent test set. Finally, we generated over 200 pseudo-bulked in silico biopsies similar in size to standard transthoracic needle biopsies using the spatial data. The scores from these in silico biopsies were similar to predictions from matched bulk RNA-seq data, suggesting robustness to intra-tumor heterogeneity. Our combined bulk and spatial transcriptomics analysis suggests that VI-associated gene expression extends far from the site of intravasation and can be used to predict the presence of VI. This may enable the prediction of angioinvasive LUAD from small biopsy specimens, allowing for more tailored treatment prior to surgery. Citation Format: Dylan Steiner, Lila Sultan, Travis Sullivan, Emily Green, Hanqiao Liu, Xiaohui Xiao, Gang Liu, Avrum Spira, Sarah Mazzilli, Kimberly Rieger-Christ, Eric Burks, Jennifer Beane, Marc Lenburg. Evaluating a novel molecular biomarker of angioinvasive lung adenocarcinoma with spatial transcriptomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5632.