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Francisella tularensis causes the severe disease tularemia. In the present study, the aim was to identify correlates of protection in the rat co-culture model by investigating the immune responses using two vaccine candidates conferring distinct degrees of protection in rat and mouse models. The immune responses were characterized by use of splenocytes from na&iuml, ve or Live vaccine strain- (LVS) or ∆clpB/∆wbtC-immunized Fischer 344 rats as effectors and bone marrow-derived macrophages infected with the highly virulent strain SCHU S4. A complex immune response was elicited, resulting in cytokine secretion, nitric oxide production, and efficient control of the intracellular bacterial growth. Addition of LVS-immune splenocytes elicited a significantly better control of bacterial growth than ∆clpB/∆wbtC splenocytes. This mirrored the efficacy of the vaccine candidates in the rat model. Lower levels of IFN-&gamma, TNF, fractalkine, IL-2, and nitrite were present in the co-cultures with ∆clpB/∆wbtC splenocytes than in those with splenocytes from LVS-immunized rats. Nitric oxide was found to be a correlate of protection, since the levels inversely correlated to the degree of protection and inhibition of nitric oxide production completely reversed the growth inhibition of SCHU S4. Overall, the results demonstrate that the co-culture assay with rat-derived cells is a suitable model to identify correlates of protection against highly virulent strains of F. tularensis

Coronavirus disease 2019 (COVID-19) is an illness caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease was first identified as a cluster of respiratory illness in Wuhan City, Hubei Province, China, in December 2019, and has rapidly spread across the globe to greater than 200 countries. Healthcare providers are at an increased risk for contracting the disease due to occupational exposure and require appropriate personal protective equipment (PPE), including N95 respirators. The rapid worldwide spread of high numbers of COVID-19 cases has facilitated the need for a substantial supply of PPE that is largely unavailable in many settings, thereby creating critical shortages. Creative solutions for the decontamination and safe reuse of PPE to protect our frontline healthcare personnel are essential. Here, we describe the development of a process that began in late February 2020 for selecting and implementing the use of hydrogen peroxide vapor (HPV) as viable method to reprocess N95 respirators. Since pre-existing HPV decontamination chambers were not available, we optimized the sterilization process in an operating room after experiencing initial challenges in other environments. Details are provided about the prioritization and implementation of processes for collection and storage, pre-processing, HPV decontamination, and post-processing of filtering facepiece respirators. Important lessons learned from this experience include, developing an adequate reserve of PPE for effective reprocessing and distribution, and identifying a suitable location with optimal environmental controls (i.e. operating room). Collectively, information presented here provides a framework for other institutions considering decontamination procedures for N95 respirators. Impact statement There is a critical shortage of personal protective equipment (PPE) around the globe. This article describes the safe collection, storage, and decontamination of N95 respirators using hydrogen peroxide vapor (HPV). This article is unique because it describes the HPV process in an operating room, and is therefore, a deployable method for many healthcare settings. Results presented here offer creative solutions to the current PPE shortage.

Bacterial persistence, known as noninherited antibacterial resistance, is a factor contributing to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate the activity of different classes of antibiotics against Burkholderia thailandensis persister cells., Bacterial persistence, known as noninherited antibacterial resistance, is a factor contributing to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate the activity of different classes of antibiotics against Burkholderia thailandensis persister cells. Here we demonstrate that addition of NA in in vitro models of B. thailandensis infection resulted in a significant depletion of the persister population in response to various classes of antibiotics. We applied microfluidic bioreactors with a continuous medium flow to study the effect of supplementation with an NA gradient on the recovery of B. thailandensis persister populations. A coculture of human neutrophils preactivated with 50 µM NA and B. thailandensis resulted in the most efficient reduction in the persister population. Applying single-cell RNA fluorescence in situ hybridization analysis and quantitative PCR, we found that NA inhibited gene expression of the stringent response regulator relA, implicated in the regulation of the persister metabolic state. We also demonstrate that a therapeutic dose of NA (250 mg/kg of body weight), previously applied as immunoprophylaxis against antibiotic-resistant bacterial species, produced adverse effects in an in vivo murine model of infection with the highly pathogenic bacterium Burkholderia pseudomallei, indicating that therapeutic dose and metabolite effects have to be carefully evaluated and tailored for every case of potential clinical application.

There are no defined correlates of protection for any intracellular pathogen, including the bacterium Francisella tularensis, which causes tularemia. Evaluating vaccine efficacy against sporadic diseases like tularemia using field trials is problematic, and therefore alternative strategies to test vaccine candidates like the Francisella Live Vaccine Strain (LVS), such as testing in animals and applying correlate measurements, are needed. Recently, we described a promising correlate strategy that predicted the degree of vaccine-induced protection in mice given parenteral challenges, primarily when using an attenuated Francisella strain. Here, we demonstrate that using peripheral blood lymphocytes (PBLs) in this approach predicts LVS-mediated protection against respiratory challenge of Fischer 344 rats with fully virulent F. tularensis, with exceptional sensitivity and specificity. Rats were vaccinated with a panel of LVS-derived vaccines and subsequently given lethal respiratory challenges with Type A F. tularensis. In parallel, PBLs from vaccinated rats were evaluated for their functional ability to control intramacrophage Francisella growth in in vitro co-culture assays. PBLs recovered from co-cultures were also evaluated for relative gene expression using a large panel of genes identified in murine studies. In vitro control of LVS intramacrophage replication reflected the hierarchy of protection. Further, despite variability between individuals, 22 genes were significantly more up-regulated in PBLs from rats vaccinated with LVS compared to those from rats vaccinated with the variant LVS-R or heat killed LVS, which were poorly protective. These genes included IFN-gamma, IL-21, NOS2, LTA, T-bet, IL-12rβ2, and CCL5. Most importantly, combining quantifications of intramacrophage growth control with 5-7 gene expression levels using multivariate analyses discriminated protected from non-protected individuals with greater than 95% sensitivity and specificity. The results therefore support translation of this approach to non-human primates and people to evaluate new vaccines against Francisella and other intracellular pathogens.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

The inbred Fischer 344 rat is being evaluated for testing novel vaccines and therapeutics against pneumonic tularemia. Although primary pneumonic tularemia in humans typically occurs by inhalation of aerosolized bacteria, the rat model has relied on intratracheal inoculation of organisms because of safety and equipment issues. We now report the natural history of pneumonic tularemia in female Fischer 344 rats after nose-only inhalational exposure to lethal doses of aerosolized Francisella tularensis subspecies tularensis, strain SCHU S4. Our results are consistent with initial uptake of aerosolized SCHU S4 from the nasal cavity, lungs, and possibly the gastrointestinal tract. Bacteremia with hematogenous dissemination was first detected 2 days after exposure. Shortly thereafter, the infected rats exhibited fever, tachypnea, and hypertension that persisted for 24 to 36 hours and then rapidly decreased as animals succumbed to infection between days 5 and 8 after exposure. Tachycardia was observed briefly, but only after the core body temperature and blood pressure began to decrease as the animals were near death. Initial neutrophilic and histiocytic inflammation in affected tissues became progressively more fibrinous and necrotizing over time. At death, as many as 1010 colony-forming units were found in the lungs, spleen, and liver. Death was attributed to sepsis and disseminated intravascular coagulation. Overall, the pathogenesis of pneumonic tularemia in the female F344 rat model appears to replicate the disease in humans.

Detection of microbial antigens in clinical samples can lead to rapid diagnosis of an infection and administration of appropriate therapeutics. A major barrier in diagnostics development is determining which of the potentially hundreds or thousands of antigens produced by a microbe are actually present in patient samples in detectable amounts against a background of innumerable host proteins. In this report, we describe a strategy, termed in vivo microbial antigen discovery (InMAD), that we used to identify circulating bacterial antigens. This technique starts with “InMAD serum,” which is filtered serum that has been harvested from BALB/c mice infected with a bacterial pathogen. The InMAD serum, which is free of whole bacterial cells, is used to immunize syngeneic BALB/c mice. The resulting “InMAD immune serum” contains antibodies specific for the soluble microbial antigens present in sera from the infected mice. The InMAD immune serum is then used to probe blots of bacterial lysates or bacterial proteome arrays. Bacterial antigens that are reactive with the InMAD immune serum are precisely the antigens to target in an antigen immunoassay. By employing InMAD, we identified multiple circulating antigens that are secreted or shed during infection using Burkholderia pseudomallei and Francisella tularensis as model organisms. Potential diagnostic targets identified by the InMAD approach included bacterial proteins, capsular polysaccharide, and lipopolysaccharide. The InMAD technique makes no assumptions other than immunogenicity and has the potential to be a broad discovery platform to identify diagnostic targets from microbial pathogens., IMPORTANCE Effective treatment of microbial infection is critically dependent on early diagnosis and identification of the etiological agent. One means for rapid diagnosis is immunoassay for antigens that are shed into body fluids during infection. Immunoassays can be inexpensive, rapid, and adaptable to a point-of-care format. A major impediment to immunoassay for diagnosis of infectious disease is identification of appropriate antigen targets. This report describes a strategy that can be used for identification of microbial antigens that are shed into serum during infection by the biothreats Burkholderia pseudomallei and Francisella tularensis. Termed InMAD (in vivo microbial antigen discovery), the strategy has the potential for application to a broad spectrum of microbial pathogens.

Pneumonic tularemia is a life-threatening disease caused by inhalation of the highly infectious intracellular bacterium Francisella tularensis . The most serious form of the disease associated with the type A strains can be prevented in experimental animals through vaccination with the attenuated live vaccine strain (LVS). The protection is largely cell mediated, but the contribution of antibodies remains controversial. We addressed this issue in a series of passive immunization studies in Fischer 344 (F344) rats. Subcutaneous LVS vaccination induced a robust serum antibody response dominated by IgM, IgG2a, and IgG2b antibodies. Prophylactic administration of LVS immune serum or purified immune IgG reduced the severity and duration of disease in naïve rats challenged intratracheally with a lethal dose of the virulent type A strain SCHU S4. The level of resistance increased with the volume of immune serum given, but the maximum survivable SCHU S4 challenge dose was at least 100-fold lower than that shown for LVS-vaccinated rats. Protection correlated with reduced systemic bacterial growth, less severe histopathology in the liver and spleen during the early phase of infection, and bacterial clearance by a T cell-dependent mechanism. Our results suggest that treatment with immune serum limited the sequelae associated with infection, thereby enabling a sterilizing T cell response to develop and resolve the infection. Thus, antibodies induced by LVS vaccination may contribute to the defense of F344 rats against respiratory infection by type A strains of F. tularensis .

Mycobacterium ulcerans (MU) is the third common mycobacterial infection after tuberculosis and leprosy. In endemic areas, MU ulcers should be considered in the differential diagnosis of any unusual or nonhealing lesion or ulcer. Diagnosis and treatment should be instigated promptly. Delay may lead to disfiguring or disabling scars. Surgical management, therefore, should aim towards early excision, with clear margins of the ulcer. We present 4 consecutive patients treated by our department within a 6-month period for MU ulcers. The presentation, diagnosis and surgical management are described. Based on our experience and after reviewing the literature, we have developed a surgical algorithm for the management of MU ulcers.

Tularemia is caused by infection with the small gram-negative coccobacillus Francisella tularensis. Pneumonic tularemia is the most serious form of tularemia and is caused by inhalation of either of the two pathogenic F. tularensis subspecies, F. tularensis subsp. tularensis (biovar A) or F. tularensis subsp. holarctica (biovar B) (8). The incidence of naturally occurring pneumonic tularemia is relatively low and is generally associated with activities such as farming and gardening that cause F. tularensis to be aerosolized and dispersed. However, the potential that F. tularensis may be prepared as a bioweapon and intentionally released as an aerosol to cause high mortality rates in the general public has led the Centers for Disease Control and Prevention of the U.S. government to list it as a category A bioterrorism agent. F. tularensis subsp. tularensis is the more virulent of the two pathogenic F. tularensis subspecies and has been estimated to cause >90% of the detected tularemia cases in North America. It is thought that the inhalation of fewer than 25 CFU of biovar A bacteria causes fulminant disease in humans (31, 34). The disease appears abruptly 3 to 5 days after exposure and can rapidly progress to severe pneumonia, respiratory failure, and death (32). Antibiotic treatments are highly effective but must be initiated early in disease development. However, diagnosis and therapeutic intervention are often delayed because of the variable clinical features of pneumonic tularemia. Preventative vaccination appears to be the best approach against pneumonic tularemia. However, there is no vaccine currently available that has been shown to completely protect humans from this disease. There is also very little information from vaccine studies of animal models to guide future vaccine development. The few vaccine studies to date have examined only the protective effects of subcutaneous (s.c.) vaccination with the highly attenuated live vaccine strain (LVS). A small study with 18 human volunteers showed that vaccination by scarification with LVS does not provide complete protection against aerosol challenge with the biovar A strain SCHU S4, and two of the vaccinated volunteers developed symptoms as severe as those in volunteers without vaccination (31). This result suggests that vaccination by scarification will likely have an unacceptably high rate of failure to protect against respiratory infections by virulent F. tularensis. The success rate may be improved by delivering the vaccine directly into the respiratory tract, like vaccines against other respiratory pathogens such as influenza virus (28) and Mycobacterium tuberculosis (2, 22). In fact, aerogenic LVS vaccination protected guinea pigs and monkeys better than s.c. LVS vaccination against respiratory infection by the biovar A strain SCHU S5 (11). However, these studies were limited in size and detail because of the animal models used, and therefore, additional studies will be required to establish the effectiveness of vaccination by the respiratory route. Mouse models are ideal for evaluating the protective effects generated by respiratory vaccination and for quickly dissecting the pulmonary immune response mediating protection. Inbred mouse strains such as BALB/c and C57BL/6 mice frequently respond differently to respiratory infections (18, 20, 24, 26, 35) and can be used to determine the mechanisms and genetic bases of resistance and susceptibility. Mouse models are also more useful for detailed analyses of the pulmonary immune responses to respiratory infections than other animal models because of the availability of transgenic and knockout mouse strains as well as analytical tools. However, mice are highly susceptible to intranasal (i.n.) (14) and aerogenic (16) LVS infections, with estimated 50% lethal dose (LD50) values of 1 × 102 and 1.5 × 103 CFU, respectively, and no murine model has been developed thus far to evaluate the protective effects of vaccination through these routes. Most studies have instead vaccinated mice intradermally (i.d.) or s.c. Two of these studies specifically examined protection against an aerosol challenge with biovar A strains and obtained conflicting results: Hodge et al. found that s.c. vaccination protected mice against SCHU S5 for at least 30 days (16), whereas Chen et al. reported that i.d. vaccination failed to protect mice against biovar A strain 33 (5). These inconsistencies highlight the need to further investigate not only the protective effects generated by s.c. or i.d. vaccination but also those generated by respiratory vaccination. A more extensive understanding of pulmonary immunity against F. tularensis biovar A will greatly improve new vaccine development. The purpose of the present study was to determine whether i.n. LVS vaccination induces effective protection in mice against an i.n. challenge with F. tularensis biovar A strains. We report that i.n. vaccination generated T-cell-dependent immunity in BALB/c mice but not in C57BL/6 mice which provided substantial resistance to i.n. and s.c. challenges with an F. tularensis biovar A strain.

While it has been known for decades that the growth of tumor transplants can be enhanced immunologically, the potential significance of these previous findings to the development of primary tumors and the mechanisms of tumor enhancement has remained obscure. This review will summarize recent experiments indicating that primary tumor development can be enhanced by active immunization. The evidence suggests that antibodies, B cells and CD4+ T cells can play a critical role in enhancing the development of primary, tumors, whereas endogenous interferon-gamma (IFNgamma) can counteract enhancement. Thus, we envision two possible functions of IFNgamma: (i) preventing B cell and antibody enhancement and (ii) counteracting tumor promotion independent of T and B cells.

Many cancers escape host immunity without losing tumor-specific rejection antigens or MHC class I expression. This study tracks the evolution of one such cancer that developed in a mouse following exposure to ultraviolet light. The primary autochthonous tumor was not highly malignant and was rejected when transplanted into naïve immunocompetent mice. Neoplastic cells isolated from the primary tumor were susceptible to the growth-inhibitory effects of IFNγ in vitro, but expressed very low levels of MHC I antigen and were resistant to tumor-specific T cells unless they were first exposed to IFNγ. Serial passage of the primary tumor cells in vivo led to a highly aggressive variant that caused fast-growing tumors in normal mice. In vitro, the variant tumor cells showed increased resistance to the growth-inhibitory effects of IFNγ but expressed high levels of immunoproteasomes and MHC I molecules and were susceptible to tumor-specific T cells even without prior exposure to IFNγ.

Objectives/Hypothesis: Two common complications of esophagectomy and immediate reconstruction comprise thoracic duct injury leading to chyle leak and anastomotic leakage. These can delay optimized nutrition, speech, and swallowing rehabilitation, and thus are important to identify and treat accordingly. When either chyle leak or anastomotic leak are clinically suspected, differentiation between the two can be very difficult clinically. As both complications may result in an increase in drain output once oral intake has occurred, an effective, quick, and accurate tool is required to determine whether this increase in drain output is related to an anastomotic leak or with a increase activity in chyle production. Study Design: Retrospective descriptive study. Methods: Description of the use of oral methylene blue dye as a safe, simple, and quick clinical bedside test. Results: When ingested orally, an anastomotic leak will lead to blue dye staining the neck drain output immediately (within seconds to minutes). A chyle leak may also result in blue staining of the drain output; however, this is not an immediate phenomenon, and rather, based on the bioavailability of methylene blue this would take a minimum of 1 hour, and more likely up to 4 hours, as the dye is absorbed into mesenteric lymphatics and travels via the thoracic duct. Conclusions: With no documented contraindications or side effects from its oral use (in the absence of hypersensitivity reactions), methylene blue is an inexpensive and freely available test in the postoperative setting of esophageal reconstruction. Laryngoscope, 2012

Background: The pathogenesis of Francisella tularensis, the causative agent of tularemia, has been primarily characterized in mice. However, the high degree of sensitivity of mice to bacterial challenge, especially with the human virulent strains of F. tularensis, limits this animal model for screening of defined attenuated vaccine candidates for protection studies. Methods and Findings: We analyzed the susceptibility of the Fischer 344 rat to pulmonary (intratracheal) challenge with three different subspecies (subsp) of F. tularensis that reflect different levels of virulence in humans, and characterized the bacterial replication profile in rat bone marrow-derived macrophages (BMDM). In contrast to the mouse, Fischer 344 rats exhibit a broader range of sensitivity to pulmonary challenge with the human virulent subsp. tularensis and holarctica. Unlike mice, Fischer rats exhibited a high degree of resistance to pulmonary challenge with LVS (an attenuated derivative of subsp. holarctica) and subsp. novicida. Within BMDM, subsp. tularensis and LVS showed minimal replication, subsp. novicida showed marginal replication, and subsp. holartica replicated robustly. The limited intramacrophage replication of subsp. tularensis and novicida strains was correlated with the induction of nitric oxide production. Importantly, Fischer 344 rats that survived pulmonary infection with subsp. novicida were markedly protected against subsequent pulmonary challenge with subsp. tularensis, suggesting that subsp. novicida may be a useful platform for the development of vaccines against subsp. tularensis. Conclusions: The Fischer 344 rat exhibits similar sensitivity to F. tularensis strains as that reported for humans, and thus the Fischer 344 ray may serve as a better animal model for tularemia vaccine development.

Acute viral and bacterial infections in the lower respiratory tract are major causes of morbidity and mortality worldwide. The proper study of pulmonary infections requires interdisciplinary collaboration among physicians and biomedical scientists to develop rational hypotheses based on clinical studies and to test these hypotheses in relevant animal models. Animal models for common lung infections are essential to understand pathogenic mechanisms and to clarify general mechanisms for host protection in pulmonary infections, as well as to develop vaccines and therapeutics. Animal models for uncommon pulmonary infections, such as those that can be caused by category A biothreat agents, are also very important because the infrequency of these infections in humans limits in-depth clinical studies. This review summarizes our understanding of innate and adaptive immune mechanisms in the lower respiratory tract and discusses how animal models for selected pulmonary pathogens can contribute to our understanding of the pathogenesis of lung infections and to the search for new vaccines and therapies.

Parenteral and respiratory vaccinations with the intracellular bacteriumFrancisella tularensishave been studied using the live vaccine strain (LVS) in a mouse model, and spleen cells from immune mice are often used for immunological studies. However, mechanisms of host immunological responses may be different in nonlymphoid organs that are important sites of infection, such as lung and liver. Using parenteral (intradermal) or respiratory (cloud aerosol) vaccination, here we examine the functions of resulting LVS-immune liver or lung cells, respectively. Surprisingly, LVS was considerably more virulent when administered by cloud aerosol than by intranasal instillation, suggesting method-dependent differences in initial localization and/or dissemination patterns. Only low doses were sublethal, and resolution of sublethal cloud aerosol infection was dependent on gamma interferon (IFN-γ), tumor necrosis factor alpha, and inducible nitric oxide synthase. Nonetheless, survival of cloud aerosol or parenteral infection resulted in the development of a protective immune response against lethal LVS intraperitoneal or aerosol challenge, reflecting development of systemic secondary immunity in both cases. Such immunity was further detected by directly examining the functions of LVS-immune lung or liver lymphocytes in vitro. Lung lymphocytes primed by respiratory infection, as well as liver lymphocytes primed by parenteral infection, clearly controlled in vitro intracellular bacterial growth primarily via mechanisms that were not dependent on IFN-γ activity. Thus, our results indicate functional similarities between immune T cells residing in spleens, livers, and lungs of LVS-immune mice.

Pneumonic tularemia caused by inhalation of the type A strains of Francisella tularensis is associated with high morbidity and mortality in humans. The only vaccine known to protect humans against this disease is the attenuated live vaccine strain (LVS), but it is not currently registered for human use. To develop a new generation of vaccines, multiple animal models are needed that reproduce the human response to F. tularensis infection and vaccination. We examined the potential use of Fischer 344 rat as such a model. Fischer 344 rats were very sensitive to intratracheal infection with the virulent type A strain SCHU S4 and generally succumbed less than 2 weeks after infection. Similar to humans and non-human primates, Fischer 344 rats vaccinated with LVS by subcutaneous or intradermal routes were protected against a greater range of respiratory SCHU S4 challenge doses than has been reported for LVS vaccinated mice. Intratracheal LVS vaccination also induced effective immunity, but it was less protective when the challenge dose exceeded 10(5) SCHU S4. LVS vaccination did not prevent SCHU S4 infection but rather controlled bacterial growth and pathology, leading to the eventual clearance of the bacteria. Our results suggest that the Fischer 344 rat may be a good model for studying pneumonic tularemia and evaluating potential vaccine candidates.

The increased incidence of emerging infections has caused a resurgence in the development of animal models in order to study their pathophysiology and develop therapeutics against them. Optimizing these models and improving our ability to extrapolate information from animals to humans is critical because in many cases the animal model will represent the only modality for efficacy testing. Francisella tularensis (F. tularensis) is an emerging pathogen that fits this category. While there is a significant body of literature that has examined infections with F. tularensis in a variety of species, the optimal small animal model has yet to be defined. A vast majority of studies have used two strains of F. tularensis, the more virulent type A strain commonly found in North America and the less virulent type B strain common to Europe. None of the small animal models described in the literature thus far behave in a fashion identical to humans with respect to their sensitivity to SCHU S4 (type A) or live vaccine strains (LVS) (attenuated type B) and an ability of LVS vaccination to consistently protect against a SCHU S4 aerosol challenge, suggesting that significant work on animal model development still remains. This report briefly describes the parameters important for animal model development and reviews the literature related to animal models of F. tularensis, including the human model, and the characterization performed for those models.

Tumor cells engineered to secrete TNF were used as a model to examine how persistently high local concentrations of TNF suppress tumor growth. TNF secretion had no effect on tumor cell proliferation in vitro but caused a very impressive growth arrest in vivo that was dependent on both bone marrow- and non-bone marrow-derived host cells expressing TNFR. Suppression also required an endogenous IFN-γ pathway consisting minimally of IFN-γ, IFN-γ receptor, Stat1, and IFN regulatory factor 1 since mice with targeted disruption of any of the four genes failed to arrest tumor growth. The ability of these mice to suppress tumor growth was restored after they were reconstituted with bone marrow cells from Wt mice. Interestingly, mice lacking the major IFN-γ-inducing cytokines IL-12 and IL-18 or T cells, B cells, and the majority of NK cells that are potential sources of IFN-γ nevertheless inhibited tumor development. Moreover, multiple lines of evidence indicated that local release of IFN-γ was not required to inhibit tumor formation. These results strongly suggest a novel function for the endogenous IFN-γ pathway that without measurable IFN-γ production or activity affects the ability of TNF to suppress tumor development.

Cancers in mouse and man express multiple tumor-specific as well as tumor-associated antigens. Immunodominance in the host response to these antigens can result in successive selection of heritable antigen loss variants. Immunodominance may also prevent the development of responses to new tumor-specific antigens that may arise during tumor progression. Some tumor-specific antigens are retained during tumor progression possibly because they are essential for survival of the malignant phenotype. Immunodominance may allow cancer cells to escape even after loss of a single MHC Class I allele because cross-presentation of the retained antigen by this allele that must be expressed on the surrounding antigen presenting cells sustains the immunodominant response. This prevents effective responses to secondary antigens that may remain as potential targets. Immunization with in vitro selected cancer cell variants that lack the immunodominant antigen can break the immunodominance and prevent escape of cancers from host immunity.

Neutron beams for neutron capture therapy (NCT) have been studied by a number of investigators,1,2,3 using high flux research reactors as the source of neutrons. There are very few research reactors in the world, probably less than ten, that can meet the conditions of both flux intensity and neutron energy spectrum. Hence the availability of NCT for patients with malignant brain tumors would be quite limited, even if all of these reactors would be able to accommodate the necessary beam design and hospital room atmosphere into the operational program.