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Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines.ImportanceMelioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS ΔcapB/iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1-3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 107 CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 107 CFU rLVS provided 100% protection, and a single dose of 107 CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS ΔcapB/iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

Inhibitory interneurons are crucial to brain function and their dysfunction is implicated in neuropsychiatric conditions. Emerging evidence indicates that cholecystokinin (CCK)-expressing interneurons (CCK+) are highly heterogenous. We find that a large subset of parvalbumin-expressing (PV+) interneurons express CCK strongly; between 40 and 56% of PV+ interneurons in mouse hippocampal CA1 express CCK. Primate interneurons also exhibit substantial PV/CCK co-expression. Mouse PV+/CCK+ and PV+/CCK- cells show distinguishable electrophysiological and molecular characteristics. Analysis of single nuclei RNA-seq and ATAC-seq data shows that PV+/CCK+ cells are a subset of PV+ cells, not of synuclein gamma positive (SNCG+) cells, and that they strongly express oxidative phosphorylation (OXPHOS) genes. We find that mitochondrial complex I and IV-associated OXPHOS gene expression is strongly correlated with CCK expression in PV+ interneurons at both the transcriptomic and protein levels. Both PV+ interneurons and dysregulation of OXPHOS processes are implicated in neuropsychiatric conditions, including autism spectrum (ASD) disorder and schizophrenia (SCZ). Analysis of human brain samples from patients with these conditions shows alterations in OXPHOS gene expression. Together these data reveal important molecular characteristics of PV-CCK co-expressing interneurons and support their implication in neuropsychiatric conditions.

To understand the neural basis of human speech control, extensive research has been done using a variety of methodologies in a range of experimental models. Nevertheless, several critical questions about learned vocal motor control still remain open. One of them is the mechanism(s) by which neurotransmitters, such as dopamine, modulate speech and song production. In this review, we bring together the two fields of investigations of dopamine action on voice control in humans and songbirds, who share similar behavioral and neural mechanisms for speech and song production. While human studies investigating the role of dopamine in speech control are limited to reports in neurological patients, research on dopaminergic modulation of bird song control has recently expanded our views on how this system might be organized. We discuss the parallels between bird song and human speech from the perspective of dopaminergic control as well as outline important differences between these species. (Contains 4 figures.)

Mycobacterium tuberculosis infects approximately one-third of the world's population, causing active tuberculosis (TB) in ~10 million people and death in ~1.5 million people annually. A potent vaccine is needed to boost the level of immunity conferred by the current Mycobacterium bovis BCG vaccine that provides moderate protection against childhood TB but variable protection against adult pulmonary TB. Previously, we developed a recombinant attenuated Listeria monocytogenes (rLm)-vectored M. tuberculosis vaccine expressing the M. tuberculosis 30-kDa major secretory protein (r30/Ag85B), recombinant attenuated L. monocytogenes ΔactA ΔinlB prfA*30 (rLm30), and showed that boosting BCG-primed mice and guinea pigs with rLm30 enhances immunoprotection against challenge with aerosolized M. tuberculosis Erdman strain. To broaden the antigen repertoire and robustness of rLm30, we constructed 16 recombinant attenuated L. monocytogenes vaccine candidates expressing 3, 4, or 5 among 15 selected M. tuberculosis antigens, verified their protein expression, genetic stability, and growth kinetics in macrophages, and evaluated them for capacity to boost protective efficacy in BCG-primed mice. We found that boosting BCG-primed C57BL/6 and BALB/c mice with recombinant attenuated L. monocytogenes multiantigenic M. tuberculosis vaccines, especially the rLm5Ag(30) vaccine expressing a fusion protein of 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and r30, enhances BCG-induced protective immunity against M. tuberculosis aerosol challenge. In immunogenicity studies, rLm5Ag(30) strongly boosts M. tuberculosis antigen-specific CD4-positive (CD4+) and CD8+ T cell-mediated TH1-type immune responses in the spleens and lungs of BCG-primed C57BL/6 mice but does so only weakly in BCG-primed BALB/c mice. Hence, rLm5Ag(30) boosts BCG-primed immunoprotection against M. tuberculosis aerosol challenge in both C57BL/6 and BALB/c mice despite major differences in the magnitude of the vaccine-induced Th1 response in these mouse strains. Given the consistency with which recombinant attenuated L. monocytogenes vaccines expressing the 5 M. tuberculosis antigens in rLm5Ag(30) are able to boost the already high level of protection conferred by BCG alone in two rigorous mouse models of pulmonary TB and the broad CD4+ and CD8+ T cell immunity induced by rLm5Ag(30), this vaccine holds considerable promise as a new vaccine to combat the TB pandemic, especially for the majority of the world's population immunized with BCG in infancy. IMPORTANCE TB, one of the world's most important infectious diseases, afflicts approximately 10 million people and kills approximately 1.5 million people annually. The current vaccine, BCG, developed over a century ago, has been administered to about 5 billion people, mostly in infancy, but is only modestly protective. Hence, a vaccine is urgently needed to boost the level of protection afforded by BCG. Herein, we describe a safe potent live vaccine that utilizes as a vector an attenuated strain of Listeria monocytogenes, a bacterium that mimics the intracellular lifestyle of Mycobacterium tuberculosis, the causative agent of TB. The vaccine produces multiple immunologically protective proteins of M. tuberculosis. In two mouse models of pulmonary TB, the vaccine boosts the level of protection afforded by BCG. Thus, this vaccine holds considerable promise as a new vaccine to combat the TB pandemic, especially for the majority of the world's population immunized with BCG.

Mycobacterium tuberculosis (Mtb) infects one-third of the world's population and is a leading cause of death from a single infectious agent. New TB vaccines are urgently needed to augment immunity conferred by the current modestly protective BCG vaccine. We have developed live attenuated recombinant Listeria monocytogenes (rLm)-vectored TB vaccines expressing five [Mpt64/23.5-EsxH/TB10.4-EsxA/ESAT6-EsxB/CFP10-Ag85B/r30] (rLmMtb5Ag) or nine (additionally EsxN-PPE68-EspA-TB8.4) immunoprotective Mtb antigens (rLmMtb9Ag) and evaluated them for safety, immunogenicity and efficacy as standalone vaccines in two mouse models and an outbred guinea pig model. In immunogenicity studies, rLmMtb5Ag administered subcutaneously induces significantly enhanced antigen-specific CD4+ and CD8+ T-cell responses in C57BL/6 and BALB/c mice, and rLmMtb9Ag induces antigen-specific CD4+ and CD8+ T-cell proliferation in guinea pigs. In efficacy studies, both rLmMtb5Ag and rLmMtb9Ag are safe and protect C57BL/6 and BALB/c mice and guinea pigs against aerosol challenge with highly virulent Mtb. Hence, multi-antigenic rLm vaccines hold promise as new vaccines against TB.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 T cell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 T cell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.

Viral tracers are important tools for neuroanatomical mapping and genetic payload delivery. Genetically modified viruses allow for cell-type-specific targeting and overcome many limitations of non-viral tracers. Here, we summarize the viruses that have been developed for neural circuit mapping, and we provide a primer on currently applied anterograde and retrograde viral tracers with practical guidance on experimental uses. We also discuss and highlight key technical and conceptual considerations for developing new safer and more effective anterograde trans-synaptic viral vectors for neural circuit analysis in multiple species.

We recently reported that the treatment with nanoparticles (NPs) loaded with tolerogenic cytokines suppressed the manifestations of lupus-like disease induced by the transfer of donor CD4+ T cells from DBA/2 mice into (C57BL/6 × DBA/2)F1 (BDF1) mice. Although the protective effects were ascribed to the induction of adaptive CD4+ and CD8+ T regulatory cells, the results suggested that another population of immune cells could be involved. Here we report that NK cells critically contribute to the protection from lupus-like disease conferred by NPs to BDF1 mice, and that this effect is TGF-β-dependent.

During homeostasis, interactions between tolerogenic dendritic cells (DCs), self-reactive T cells, and T regulatory cells (Tregs) contribute to maintaining mammalian immune tolerance. In response to infection, immunogenic DCs promote the generation of proinflammatory effector T cell subsets. When complex homeostatic mechanisms maintaining the balance between regulatory and effector functions become impaired, autoimmune diseases can develop. We discuss some of the newest advances on the mechanisms of physiopathologic homeostasis that can be employed to develop strategies to restore a dysregulated immune equilibrium. Some of these designs are based on selectively activating regulators of immunity and inflammation instead of broadly suppressing these processes. Promising approaches include the use of nanoparticles (NPs) to restore Treg control over self-reactive cells, aiming to achieve long-term disease remission, and potentially to prevent autoimmunity in susceptible individuals.

Mesoporous silica nanoparticles (MSNs) are delivery vehicles that can carry cargo molecules and release them on command. The particles used in the applications reported in this Account are around 100 nm in diameter (about the size of a virus) and contain 2.5 nm tubular pores with a total volume of about 1 cm3/g. For the biomedical applications discussed here, the cargo is trapped in the pores until the particles are stimulated to release it. The challenges are to get the particles to the site of a disease and then to deliver the cargo on command. We describe methods to do both, and we illustrate the applicability of the particles to cure cancer and intracellular infectious disease. Our first steps were to design multifunctional nanoparticles with properties that allow them to carry and deliver hydrophobic drugs. Many important pharmaceuticals are hydrophobic and cannot reach the diseased sites by themselves. We describe how we modified MSNs to make them dispersible, imagable, and targetable and discuss in vitro studies. We then present examples of surface modifications that allow them to deliver large molecules such as siRNA. In vivo studies of siRNA delivery to treat triple-negative breast and ovarian cancers are presented. The next steps are to attach nanomachines and other types of caps that trap drug molecules but release them when stimulated. We describe nanomachines that respond autonomously (without human intervention) to stimuli specific to disease sites. A versatile type of machine is a nanovalve that is closed at neutral (blood) pH but opens upon acidification that occurs in endolysosomes of cancer cells. Another type of machine, a snap-top cap, is stimulated by reducing agents such as glutathione in the cytosol of cells. Both of these platforms were studied in vitro to deliver antibiotics to infected macrophages and in vivo to cure and kill the intracellular bacteria M. tuberculosis and F. tularensis. The latter is a tier 1 select agent of bioterrorism. Finally, we describe nanomachines for drug delivery that are controlled by externally administered light and magnetic fields. A futuristic dream for nanotherapy is the ability to control a nano-object everywhere in the body. Magnetic fields penetrate completely and have spatial selectivity governed by the size of the field-producing coil. We describe how to control nanovalves with alternating magnetic fields (AMFs) and superparamagnetic cores inside the MSNs. The AMF heats the cores, and temperature-sensitive caps release the cargo. In vitro studies demonstrate dose control of the therapeutic to cause apoptosis without overheating the cells. Nanocarriers have great promise for therapeutic applications, and MSNs that can carry drugs to the site of a disease to produce a high local concentration without premature release and off-target damage may have the capability of realizing this goal.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

ObjectiveTo develop a nanoparticle (NP) platform that can expand both CD4+ and CD8+ Treg cells in vivo for the suppression of autoimmune responses in systemic lupus erythematosus (SLE).MethodsPoly(lactic-co-glycolic acid) (PLGA) NPs encapsulating interleukin-2 (IL-2) and transforming growth factor β (TGFβ) were coated with anti-CD2/CD4 antibodies and administered to mice with lupus-like disease induced by the transfer of DBA/2 T cells into (C57BL/6 × DBA/2)F1 (BDF1) mice. The peripheral frequency of Treg cells was monitored ex vivo by flow cytometry. Disease progression was assessed by measuring serum anti-double-stranded DNA antibody levels by enzyme-linked immunosorbent assay. Kidney disease was defined as the presence of proteinuria or renal histopathologic features.ResultsAnti-CD2/CD4 antibody-coated, but not noncoated, NPs encapsulating IL-2 and TGFβ induced CD4+ and CD8+ FoxP3+ Treg cells in vitro. The optimal dosing regimen of NPs for expansion of CD4+ and CD8+ Treg cells was determined in in vivo studies in mice without lupus and then tested in BDF1 mice with lupus. The administration of anti-CD2/CD4 antibody-coated NPs encapsulating IL-2 and TGFβ resulted in the expansion of CD4+ and CD8+ Treg cells, a marked suppression of anti-DNA antibody production, and reduced renal disease.ConclusionThis study shows for the first time that T cell-targeted PLGA NPs encapsulating IL-2 and TGFβ can expand both CD4+ and CD8+ Treg cells in vivo and suppress murine lupus. This approach, which enables the expansion of Treg cells in vivo and inhibits pathogenic immune responses in SLE, could represent a potential new therapeutic modality in autoimmune conditions characterized by impaired Treg cell function associated with IL-2 deficiency.

Francisella tularensis causes a serious and often fatal infection, tularemia. We compared the efficacy of moxifloxacin formulated as free drug vs disulfide snap-top mesoporous silica nanoparticles (MSNs) in a mouse model of pneumonic tularemia. We found that MSN-formulated moxifloxacin was more effective than free drug and that the intramuscular and subcutaneous routes were markedly more effective than the intravenous route. Measurement of tissue silica levels and fluorescent flow cytometry assessment of colocalization of MSNs with infected cells revealed that the enhanced efficacy of MSNs and the intramuscular route of delivery was not due to better delivery of MSNs to infected tissues or cells. However, moxifloxacin blood levels demonstrated that the nanoparticle formulation and intramuscular route provided the longest half-life and longest time above the minimal inhibitory concentration. Thus, improved pharmacokinetics are responsible for the greater efficacy of nanoparticle formulation and intramuscular delivery compared with free drug and intravenous delivery.

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

BackgroundShorter, more effective treatments for tuberculosis (TB) are urgently needed. While many TB drugs are available, identification of the best regimens is challenging because of the large number of possible drug-dose combinations. We have found consistently that responses of cells or whole animals to drug-dose stimulations fit a parabolic response surface (PRS), allowing us to identify and optimize the best drug combinations by testing only a small fraction of the total search space. Previously, we used PRS methodology to identify three regimens (PRS Regimens I-III) that in murine models are much more effective than the standard regimen used to treat TB. However, PRS Regimens I and II are unsuitable for treating drug-resistant TB and PRS Regimen III includes an experimental drug. Here, we use PRS methodology to identify from an expanded pool of drugs new highly effective near-universal drug regimens comprising only approved drugs.Methods and findingsWe evaluated combinations of 15 different drugs in a human macrophage TB model and identified the most promising 4-drug combinations. We then tested 14 of these combinations in Mycobacterium tuberculosis-infected BALB/c mice and chose for PRS dose optimization and further study the two most potent regimens, designated PRS Regimens IV and V, consisting of clofazimine (CFZ), bedaquiline (BDQ), pyrazinamide (PZA), and either amoxicillin/clavulanate (AC) or delamanid (DLM), respectively. We then evaluated the efficacy in mice of optimized PRS Regimens IV and V, as well as a 3-drug regimen, PRS Regimen VI (CFZ, BDQ, and PZA), and compared their efficacy to PRS Regimen III (CFZ, BDQ, PZA, and SQ109), previously shown to reduce the time to achieve relapse-free cure in mice by 80% compared with the Standard Regimen (isoniazid, rifampicin, PZA, and ethambutol). Efficacy measurements included early bactericidal activity, time to lung sterilization, and time to relapse-free cure. PRS Regimens III-VI all rapidly sterilized the lungs and achieved relapse-free cure in 3 weeks (PRS Regimens III, V, and VI) or 5 weeks (PRS Regimen IV). In contrast, mice treated with the Standard Regimen still had high numbers of bacteria in their lungs after 6-weeks treatment and none achieved relapse-free cure in this time-period.ConclusionsWe have identified three new regimens that rapidly sterilize the lungs of mice and dramatically shorten the time required to achieve relapse-free cure. All mouse drug doses in these regimens extrapolate to doses that are readily achievable in humans. Because PRS Regimens IV and V contain only one first line drug (PZA) and exclude fluoroquinolones and aminoglycosides, they should be effective against most TB cases that are multidrug resistant (MDR-TB) and many that are extensively drug-resistant (XDR-TB). Hence, these regimens have potential to shorten dramatically the time required for treatment of both drug-sensitive and drug-resistant TB. If clinical trials confirm that these regimens dramatically shorten the time required to achieve relapse-free cure in humans, then this radically shortened treatment has the potential to improve treatment compliance, decrease the emergence of drug resistance, and decrease the healthcare burden of treating both drug-sensitive and drug-resistant TB.

Bacillus anthracis, Yersinia pestis, and Francisella tularensis are the causative agents of Tier 1 Select Agents anthrax, plague, and tularemia, respectively. Currently, there are no licensed vaccines against plague and tularemia and the licensed anthrax vaccine is suboptimal. Here we report F. tularensis LVS ΔcapB (Live Vaccine Strain with a deletion in capB)- and attenuated multi-deletional Listeria monocytogenes (Lm)-vectored vaccines against all three aforementioned pathogens. We show that LVS ΔcapB- and Lm-vectored vaccines express recombinant B. anthracis, Y. pestis, and F. tularensis immunoprotective antigens in broth and in macrophage-like cells and are non-toxic in mice. Homologous priming-boosting with the LVS ΔcapB-vectored vaccines induces potent antigen-specific humoral and T-cell-mediated immune responses and potent protective immunity against lethal respiratory challenge with all three pathogens. Protection against anthrax was far superior to that obtained with the licensed AVA vaccine and protection against tularemia was comparable to or greater than that obtained with the toxic and unlicensed LVS vaccine. Heterologous priming-boosting with LVS ΔcapB- and Lm-vectored B. anthracis and Y. pestis vaccines also induced potent protective immunity against lethal respiratory challenge with B. anthracis and Y. pestis. The single vaccine platform, especially the LVS ΔcapB-vectored vaccine platform, can be extended readily to other pathogens.

Francisella tularensisis subsp. tularensis is an intracellular bacterial pathogen and the causative agent of the life-threatening zoonotic disease tularemia. The Francisella Pathogenicity Island encodes a large secretion apparatus, known as a Type VI Secretion System (T6SS), which is essential for Francisella to escape from its phagosome and multiply within host macrophages and to cause disease in animals. The T6SS, found in one-quarter of Gram-negative bacteria including many highly pathogenic ones, is a recently discovered secretion system that is not yet fully understood. Nevertheless, there have been remarkable advances in our understanding of the structure, composition, and function of T6SSs of several bacteria in the past few years. The system operates like an inside-out headless contractile phage that is anchored to the bacterial membrane via a baseplate and membrane complex. The system injects effector molecules across the inner and outer bacterial membrane and into host prokaryotic or eukaryotic targets to kill, intoxicate, or in the case of Francisella, hijack the target cell. Recent advances include an atomic model of the contractile sheath, insights into the mechanics of sheath contraction, the composition of the baseplate and membrane complex, the process of assembly of the apparatus, and identification of numerous effector molecules and activities. While Francisella T6SS appears to be an outlier among T6SSs, with limited or no sequence homology with other systems, its structure and organization are strikingly similar to other systems. Nevertheless, we have only scratched the surface in uncovering the mysteries of the Francisella T6SS, and there are numerous questions that remain to be answered.

Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of having efficacy ≥LVS in the demanding mouse model of tularemia. These latter include LVS with deletions in purMCD, sodBFt , capB or wzy; LVS ΔcapB that also overexpresses Type VI Secretion System (T6SS) proteins; FSC200 with a deletion in clpB; the single deletional purMCD mutant of F. tularensis SCHU S4, and a heterologous prime-boost vaccine comprising LVS ΔcapB and Listeria monocytogenes expressing T6SS proteins.

As current treatment of tuberculosis is burdensomely long, provoking non-adherence and drug resistance, effective short-course treatments are needed. Using the output-driven parabolic response surface (PRS) platform, we have identified drug regimens that treat tuberculosis more rapidly in mice than the current Standard Regimen used in humans. We show that PRS Regimen III, comprising clofazimine, SQ109, bedaquiline and pyrazinamide, rapidly sterilizes the lung both in conventionally studied BALB/c mice and in C3HeB/FeJ mice, highly susceptible mice that develop massive necrotic granulomatous lung lesions akin to those in humans, achieving relapse-free cure in only 4 weeks (p

The iron-regulatory hormone hepcidin is induced early in infection, causing iron sequestration in macrophages and decreased plasma iron; this is proposed to limit the replication of extracellular microbes, but could also promote infection with macrophage-tropic pathogens. The mechanisms by which hepcidin and hypoferremia modulate host defense, and the spectrum of microbes affected, are poorly understood. Using mouse models, we show that hepcidin was selectively protective against siderophilic extracellular pathogens (Yersinia enterocolitica O9) by controlling non-transferrin-bound iron (NTBI) rather than iron-transferrin concentration. NTBI promoted the rapid growth of siderophilic but not nonsiderophilic bacteria in mice with either genetic or iatrogenic iron overload and in human plasma. Hepcidin or iron loading did not affect other key components of innate immunity, did not indiscriminately promote intracellular infections (Mycobacterium tuberculosis), and had no effect on extracellular nonsiderophilic Y enterocolitica O8 or Staphylococcus aureus Hepcidin analogs may be useful for treatment of siderophilic infections.

The current drug regimens for treating tuberculosis are lengthy and onerous, and hence complicated by poor adherence leading to drug resistance and disease relapse. Previously, using an output-driven optimization platform and an in vitro macrophage model of Mycobacterium tuberculosis infection, we identified several experimental drug regimens among billions of possible drug-dose combinations that outperform the current standard regimen. Here we use this platform to optimize the in vivo drug doses of two of these regimens in a mouse model of pulmonary tuberculosis. The experimental regimens kill M. tuberculosis much more rapidly than the standard regimen and reduce treatment time to relapse-free cure by 75%. Thus, these regimens have the potential to provide a markedly shorter course of treatment for tuberculosis in humans. As these regimens omit isoniazid, rifampicin, fluoroquinolones and injectable aminoglycosides, they would be suitable for treating many cases of multidrug and extensively drug-resistant tuberculosis.

Some HIV-1-infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual's serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1YU2-infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection.

A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ΔcapB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS ΔcapB/bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS ΔcapB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis.

Effective and rapid treatment of tularemia is needed to reduce morbidity and mortality of this potentially fatal infectious disease. The etiologic agent, Francisella tularensis, is a facultative intracellular bacterial pathogen which infects and multiplies to high numbers in macrophages. Nanotherapeutics are particularly promising for treatment of infectious diseases caused by intracellular pathogens, whose primary host cells are macrophages, because nanoparticles preferentially target and are avidly internalized by macrophages. A mesoporous silica nanoparticle (MSN) has been developed functionalized with disulfide snap-tops that has high drug loading and selectively releases drug intracellularly in response to the redox potential. These nanoparticles, when loaded with Hoechst fluorescent dye, release their cargo exclusively intracellularly and stain the nuclei of macrophages. The MSNs loaded with moxifloxacin kill F. tularensis in macrophages in a dose-dependent fashion. In a mouse model of lethal pneumonic tularemia, MSNs loaded with moxifloxacin prevent weight loss, illness, and death, markedly reduce the burden of F. tularensis in the lung, liver, and spleen, and are significantly more efficacious than an equivalent amount of free drug. An important proof-of-principle for the potential therapeutic use of a novel nanoparticle drug delivery platform for the treatment of infectious diseases is provided.

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.

Persistent viral infections are simultaneously associated with chronic inflammation and highly potent immunosuppressive programs mediated by IL-10 and PDL1 that attenuate antiviral T cell responses. Inhibiting these suppressive signals enhances T cell function to control persistent infection; yet, the underlying signals and mechanisms that program immunosuppressive cell fates and functions are not well understood. Herein, we use lymphocytic choriomeningitis virus infection (LCMV) to demonstrate that the induction and functional programming of immunosuppressive dendritic cells (DCs) during viral persistence are separable mechanisms programmed by factors primarily considered pro-inflammatory. IFNγ first induces the de novo development of naive monocytes into DCs with immunosuppressive potential. Type I interferon (IFN-I) then directly targets these newly generated DCs to program their potent T cell immunosuppressive functions while simultaneously inhibiting conventional DCs with T cell stimulating capacity. These mechanisms of monocyte conversion are constant throughout persistent infection, establishing a system to continuously interpret and shape the immunologic environment. MyD88 signaling was required for the differentiation of suppressive DCs, whereas inhibition of stimulatory DCs was dependent on MAVS signaling, demonstrating a bifurcation in the pathogen recognition pathways that promote distinct elements of IFN-I mediated immunosuppression. Further, a similar suppressive DC origin and differentiation was also observed in Mycobacterium tuberculosis infection, HIV infection and cancer. Ultimately, targeting the underlying mechanisms that induce immunosuppression could simultaneously prevent multiple suppressive signals to further restore T cell function and control persistent infections.

We have optimized mesoporous silica nanoparticles (MSNs) functionalized with pH-sensitive nanovalves for the delivery of the broad spectrum fluoroquinolone moxifloxacin (MXF) and demonstrated its efficacy in treating Francisella tularensis infections both in vitro and in vivo. We compared two different nanovalve systems, positive and negative charge modifications of the mesopores, and different loading conditions-varying pH, cargo concentration, and duration of loading-and identified conditions that maximize both the uptake and release capacity of MXF by MSNs. We have demonstrated in macrophage cell culture that the MSN-MXF delivery platform is highly effective in killing F. tularensis in infected macrophages, and in a mouse model of lethal pneumonic tularemia, we have shown that the drug-loaded MSNs are much more effective in killing F. tularensis than an equivalent amount of free MXF.

The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.

Tuberculosis is a major global health problem for which improved therapeutics are needed to shorten the course of treatment and combat emergence of drug resistance. Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of mononuclear phagocytes. As such, it is an ideal pathogen for nanotherapeutics because macrophages avidly ingest nanoparticles even without specific targeting molecules. Hence, a nanoparticle drug delivery system has the potential to target and deliver high concentrations of drug directly into M. tuberculosis-infected cells-greatly enhancing efficacy while avoiding off-target toxicities. Stimulus-responsive mesoporous silica nanoparticles of two different sizes, 100 and 50 nm, are developed as carriers for the major anti-tuberculosis drug isoniazid in a prodrug configuration. The drug is captured by the aldehyde-functionalized nanoparticle via hydrazone bond formation and coated with poly(ethylene imine)-poly(ethylene glycol) (PEI-PEG). The drug is released from the nanoparticles in response to acidic pH at levels that naturally occur within acidified endolysosomes. It is demonstrated that isoniazid-loaded PEI-PEG-coated nanoparticles are avidly ingested by M. tuberculosis-infected human macrophages and kill the intracellular bacteria in a dose-dependent manner. It is further demonstrated in a mouse model of pulmonary tuberculosis that the nanoparticles are well tolerated and much more efficacious than an equivalent amount of free drug.

On page 5066, J. I. Zink, M. A. Horwitz, and co-workers use confocal microscopy to demonstrate the avid uptake of RITC-labeled mesoporous silica nanoparticles loaded with the anti-tuberculosis drug isoniazid (shown here in red) by human macrophages (nuclei stained blue with DAPI) infected with GFP-expressing Mycobacterium tuberculosis (shown here in green).

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

BackgroundA complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.ResultsWe introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, "when" and "where" molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature.ConclusionsThese observations suggest a working model for NA assembly whereby transient α-actinin-integrin complexes help nucleate NAs within the lamellipodium. Subsequently, integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes.

β-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of β-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1β as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1β is induced by DNA damage via an NF-κB-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1β receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1β prevents epithelial tight junction dysfunction and alleviates mucositis in β-TrCP-deficient mice. IL-1β antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

Chronic iron-overload is damaging to the heart, liver, and other organs. Better iron chelators are needed to treat this serious medical condition. The uptake and distribution of the lipid-soluble, hexadentate iron chelator desferri-Exochelin 772SM (D-Exo) is studied and its efficacy in removing iron from tissue in rodent models is evaluated. After an intravenous bolus of tritiated D-Exo to rats, counts rapidly disappeared from the blood and rapidly appeared in 15 organs studied, usually peaking within 15 min. There was considerable uptake in the heart and liver, 2 organs especially susceptible to damage from clinical iron overload. To assess actual decreases in cardiac and hepatic iron in response to D-Exo, mice loaded with 42 mg of iron dextran (2100 mg/kg) were studied. Untreated, iron-loaded mice sacrificed 9 weeks later had a 4-fold increase in cardiac iron and a 20-fold increase in hepatic iron compared with controls that were not iron-loaded. In iron-loaded mice treated with 7 mg of D-Exo intraperitoneally (i.p.) 4 days/week for 8 weeks (total 224 mg), tissue iron, measured by atomic absorption, was reduced by 20% in the liver and 25% in the heart (P < 0.01 for each organ). During the first 8 h after a D-Exo dose, iron was excreted in the urine. Mice treated with D-Exo gained weight normally and showed no evidence of toxicity. In conclusion, in this iron-overload mouse model, D-Exo administered intravenously or i.p. rapidly diffuses into multiple organs, including the heart and liver, and effectively removes iron without apparent toxicity.

We previously showed that, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), vaccination with bacillus Calmette-Guerin (BCG) prior to MPTP exposure limited the loss of striatal dopamine (DA) and dopamine transporter (DAT) and prevented the activation of nigral microglia. Here, we conducted BCG dose studies and investigated the mechanisms underlying BCG vaccination's neuroprotective effects in this model. We found that a dose of 1 × 10(6) cfu BCG led to higher levels of striatal DA and DAT ligand binding (28% and 42%, respectively) in BCG-vaccinated vs. unvaccinated MPTP-treated mice, but without a significant increase in substantia nigra tyrosine hydroxylase-staining neurons. Previous studies showed that BCG can induce regulatory T cells (Tregs) and that Tregs are neuroprotective in models of neurodegenerative diseases. However, MPTP is lymphotoxic, so it was unclear whether Tregs were maintained after MPTP treatment and whether a relationship existed between Tregs and the preservation of striatal DA system integrity. We found that, 21 days post-MPTP treatment, Treg levels in mice that had received BCG prior to MPTP were threefold greater than those in MPTP-only-treated mice and elevated above those in saline-only-treated mice, suggesting that the persistent BCG infection continually promoted Treg responses. Notably, the magnitude of the Treg response correlated positively with both striatal DA levels and DAT ligand binding. Therefore, BCG vaccine-mediated neuroprotection is associated with Treg levels in this mouse model. Our results suggest that BCG-induced Tregs could provide a new adjunctive therapeutic approach to ameliorating pathology associated with PD and other neurodegenerative diseases.

Francisella tularensis, the causative agent of tularemia, is a category A bioterrorism agent. A vaccine that is safer and more effective than the currently available unlicensed F. tularensis live vaccine strain (LVS) is needed to protect against intentional release of aerosolized F. tularensis, the most dangerous type of exposure. In this study, we employed a heterologous prime-boost vaccination strategy comprising intradermally administered LVS ΔcapB (highly attenuated capB-deficient LVS mutant) as the primer vaccine and rLm/iglC (recombinant attenuated Listeria monocytogenes expressing the F. tularensis immunoprotective antigen IglC) as the booster vaccine. Boosting LVS ΔcapB-primed mice with rLm/iglC significantly enhanced T cell immunity; their splenic T cells secreted significantly more gamma interferon (IFN-γ) and had significantly more cytokine (IFN-γ and/or tumor necrosis factor [TNF] and/or interleukin-2 [IL-2])-producing CD4(+) and CD8(+) T cells upon in vitro IglC stimulation. Importantly, mice primed with LVS ΔcapB or rLVS ΔcapB/IglC, boosted with rLm/iglC, and subsequently challenged with 10 50% lethal doses (LD50) of aerosolized highly virulent F. tularensis Schu S4 had a significantly higher survival rate and mean survival time than mice immunized with only LVS ΔcapB (P < 0.0001); moreover, compared with mice immunized once with LVS, primed-boosted mice had a higher survival rate (75% versus 62.5%) and mean survival time during the first 21 days postchallenge (19 and 20 days for mice boosted after being primed with LVS ΔcapB and rLVS ΔcapB/IglC, respectively, versus 17 days for mice immunized with LVS) and maintained their weight significantly better (P < 0.01). Thus, the LVS ΔcapB-rLm/iglC prime-boost vaccination strategy holds substantial promise for a vaccine that is safer and at least as potent as LVS.

PurposeThe roles that crystallin proteins play during lens development are not well understood. Similarities in the adult crystallin composition of mammalian and zebrafish lenses have made the latter a valuable model for examining lens function. In this study, we describe the changing zebrafish lens proteome during development to identify ontogenetic shifts in crystallin expression that may provide insights into age-specific functions.MethodsTwo-dimensional gel electrophoresis and size exclusion chromatography were used to characterize the lens crystallin content of 4.5-day to 27-month-old zebrafish. Protein spots were identified with mass spectrometry and comparisons with previously published proteomic maps, and quantified with densitometry. Constituents of size exclusion chromatography elution peaks were identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.ResultsZebrafish lens crystallins were expressed in three ontogenetic patterns, with some crystallins produced at relatively constant levels throughout development, others expressed primarily before 10 weeks of age (βB1-, βA1-, and γN2-crystallins), and a third group primarily after 10 weeks (α-, βB3-, and γS-crystallins). Alpha-crystallins comprised less than 1% of total lens protein in 4.5-day lenses and increased to less than 7% in adult lenses. The developmental period between 6 weeks and 4 months contained the most dramatic shifts in lens crystallin expression.ConclusionsThese data provide the first two-dimensional gel electrophoresis maps of the developing zebrafish lens, with quantification of changing crystallin abundance and visualization of post-translational modification. Results suggest that some crystallins may play stage specific roles during lens development. The low levels of zebrafish lens α-crystallin relative to mammals may be due to the high concentrations of γ-crystallins in this aquatic lens. Similarities with mammalian crystallin expression continue to support the use of the zebrafish as a model for lens crystallin function.

Ischemia/reperfusion (I/R) injury is a serious potential threat to outcomes in organ transplantation and other clinical arenas in which there is temporary interruption of blood flow. I/R is a frequent cause of primary failure in organ transplantation. We hypothesized that the antioxidant bucillamine, a potent sulfhydryl donor, would protect against I/R injury in high-risk organ transplants. Because livers subjected to prolonged ischemia and very fatty livers are highly susceptible to severe I/R injury, we studied the effect of bucillamine in three animal models of liver transplantation: two ex vivo models of isolated perfused livers, either normal or fatty rat livers, and an in vivo model of syngenic orthotopic liver transplants in rats. In all models, livers were deprived of oxygen for 24 h before either ex vivo reperfusion or transplantation. In the ex vivo models, bucillamine treatment significantly improved portal vein blood flow and bile production, preserved normal liver architecture, and significantly reduced liver enzyme release and indices of oxidative stress. Moreover, bucillamine treatment significantly increased levels of reduced glutathione in the liver and lowered levels of oxidized glutathione in both liver and blood. In rats subjected to liver transplants, bucillamine significantly enhanced survival and protected against hepatic injury. Possible mechanisms of this protection include prevention of excessive accumulation of toxic oxygen species, interruption of redox signaling in hepatocytes, and inhibition of macrophage activation. This study demonstrates the potential utility of bucillamine or other cysteine-derived thiol donors for improving outcomes in organ transplantation and other clinical settings involving I/R injury.

Although several epidemiological studies indicate a correlation between exposure to ambient particulate matter and adverse health effects in humans, there is still a fundamental lack of understanding of the mechanisms involved. We set out to test the hypothesis that reactive oxygen species are involved in the adjuvant effects of diesel exhaust particles (DEP) in a murine OVA sensitization model. First, we tested six different antioxidants, N-acetylcysteine (NAC), bucillamine (BUC), silibinin, luteolin, trolox (vitamin E), and ascorbic acid, for their ability to interfere in DEP-mediated oxidative stress in vitro. Of the six agents tested, only the thiol antioxidants, BUC and NAC, were effective at preventing a decrease in intracellular reduced glutathione:glutathione disulfide ratios, protecting cells from protein and lipid oxidation, and preventing heme oxygenase 1 expression. Therefore, we selected the thiol antioxidants for testing in the murine OVA inhalation sensitization model. Our data demonstrate that NAC and BUC effectively inhibited the adjuvant effects of DEP in the induction of OVA-specific IgE and IgG1 production. Furthermore, NAC and BUC prevented the generation of lipid peroxidation and protein oxidation in the lungs of OVA- plus DEP-exposed animals. These findings indicate that NAC and BUC are capable of preventing the adjuvant effects of inhaled DEP and suggest that oxidative stress is a key mechanistic component in the adjuvant effect of DEP. Antioxidant treatment strategies may therefore serve to alleviate allergic inflammation and may provide a rational basis for treating the contribution of particulate matter to asthmatic disease.

Background- Vascular smooth muscle cell (VSMC) proliferation is a critical factor in the neointima formation that causes restenosis after coronary angioplasty (PTCA). Desferri-exochelin 772SM (D-EXO), a highly diffusible, lipophilic iron chelator secreted by Mycobacterium tuberculosis, inhibits proliferation of VSMCs in culture. We hypothesized that treatment with D-EXO would inhibit neointima formation in balloon-injured vessels in vivo. Methods and Results- We subjected 24 pigs to overstretch coronary artery injury with standard PTCA balloons and then administered intramural injections of either D-EXO (n=14) or vehicle (n=10) through an Infiltrator catheter. Treatments were randomized, and the investigators were blinded with regard to treatment group until data analysis was completed. One month later, we euthanized the pigs, excised the injured coronary segments, made multiple sections of each segment, and identified the site of maximal neointima formation. An injury score based on the degree of disruption of the internal or external elastic lamina or media was assigned. D-EXO reduced stenosis index (neointima area divided by the area within the internal elastic lamina), adjusted for injury score, by 47%. Neointima thickness was also reduced. Conclusions- D-EXO, injected intramurally, substantially inhibited formation of neointima in a porcine vascular injury model.

BackgroundHepatic ischemia/reperfusion (I/R) injury is associated with early and late graft failure after liver transplantation. A major mechanism is leukocyte adhesion to endothelium followed by release of reactive oxygen intermediates. We examined whether desferriexochelin 772SM (D-Exo), a lipid soluble iron chelator that prevents hydroxyl radical formation, can enhance the capacity of recombinant P-selectin glycoprotein ligand immunoglobulin (rPSGL-Ig), a glycoprotein that binds to P-selectin and inhibits neutrophil adhesion, to protect against I/R injury in an ex vivo rat liver model.MethodsRat livers were harvested and stored for 6 hr at 4 degrees C in University of Wisconsin solution and then perfused with oxygenated whole blood for 2 hr. Three groups were studied (n=6 rats/group): an untreated control group; a group that received 0.4 mg/kg rPSGL-Ig intraportally at the time of harvest; and a group that received 0.4 mg/kg rPSGL-Ig plus 1 micromol D-Exo intraportally both at the time of harvest and at the onset of reperfusion. Liver portal venous blood flow was assessed during perfusion, and at the end of each experiment, liver samples were collected for blinded histological evaluation and biochemical analyses.ResultsLivers treated with D-Exo + rPSGL-Ig had significantly higher blood flow than livers treated with rPSGL-1Ig alone (P

Cell-mediated adaptive immunity is critical for host defense, but little is known about T cell behavior during delivery of effector function. Here we investigate relationships among antigen presentation, T cell motility, and local production of effector cytokines by CD4+ T cells within hepatic granulomas triggered by Bacille Calmette-Guérin or Mycobacterium tuberculosis. At steady-state, only small fractions of mycobacteria-specific T cells showed antigen-induced migration arrest within granulomas, resulting in low-level, polarized secretion of cytokines. However, exogenous antigen elicited rapid arrest and robust cytokine production by the vast majority of effector T cells. These findings suggest that limited antigen presentation and/or recognition within granulomas evoke a muted T cell response drawing on only a fraction of the host's potential effector capacity. Our results provide new insights into the regulation of host-protective functions, especially how antigen availability influences T cell dynamics and, in turn, effector T cell function during chronic infection.

We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS rearranged methyltransferase2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA-directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term domains rearranged methyltransferase3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA-directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA-directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA-directed DNA methylation systems consist of a combination of ancestral and convergent features.

Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ΔcapB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ΔcapB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ΔcapB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ΔcapB is significantly attenuated in mice; the 50% lethal dose (LD(50)) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ΔcapB partially restores its virulence in mice. Mice immunized with LVS ΔcapB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ΔcapB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10× the LD(50) of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ΔcapB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.

We describe a method to detect molecular complexes and measure their stoichiometry in living cells from simultaneous fluctuations of the fluorescence intensity in two image channels, each detecting a different kind of protein. The number and brightness (N&B) analysis, namely, the use of the ratio between the variance and the average intensity to obtain the brightness of molecules, is extended to the cross-variance of the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or focal adhesion kinase (FAK) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin, and paxillin-tagged with EGFP and mCherry. We found no complexes of these proteins in the cytoplasm away from the adhesions. However, at the adhesions, large aggregates leave, forming a hole, during their disassembly. This hole shows cross-correlation between FAK and paxillin and vinculin and paxillin. From the amplitude of the correlated fluctuations we determine the composition of the aggregates leaving the adhesions. These aggregates disassemble rapidly in the cytoplasm because large complexes are found only in very close proximity to the adhesions or at their borders.

Fransicella tularensis, the causative agent of tularemia, is in the top category (Category A) of potential agents of bioterrorism. To develop a safer vaccine against aerosolized F. tularensis, we have employed an attenuated Listeria monocytogenes, which shares with F. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for F. tularensis antigens. We constructed recombinant L. monocytogenes (rLm) vaccines stably expressing seven F. tularensis proteins including IglC (rLm/iglC), and tested their immunogenicity and protective efficacy against lethal F. tularensis challenge in mice. Mice immunized intradermally with rLm/iglC developed significant cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon gamma. Moreover, mice immunized with rLm/iglC were protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain.