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.