The rLVS ΔcapB/iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains.

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.

IL-6 Enhances the Viability and Invasion Ability of Prostate Cancer Cells.

Prostate cancer is a slow-growing cancer whose incidence increases with age. IL-6 expression is significantly increased in a variety of malignant tumor stroma, which is a key factor involved in the association between inflammation and tumor. So as to investigate how prostate cancer works, recombinant IL-6 was used to stimulate human prostate cancer cells (PC-3). After treatment for 24,48,72h, the viability of cancer cells was detected by CCK-8 experiment. The effect of recombinant IL-6 on the invasion of prostate cancer cells was analyzed by Transwell experiment. The results showed that IL-6 can increase the viability of prostate cancer cells and facilitate the invasion of prostate cancer cells.

TRPM2-L Participates in the Interleukin-6 Pathway to Enhance Tumor Growth in Prostate Cancer by Hypoxia-Inducible Factor-1α.

Interleukin-6 (IL-6) can promote cell proliferation in prostate cancer (PCa). Full-length transient receptor potential melastatin 2 (TRPM2-L) is highly expressed in PCa. However, the association between IL-6 and TRPM2-L in PCa is unclear. Here, human PCa cell lines, PC-3 and DU-145, were treated with 10 µg/mL tocilizumab, an IL-6 receptor (IL-6R) inhibitor, and the TRPM2-L protein expression in cells was significantly decreased. Cells were stably transfected with TRPM2 short-interfering RNA (siRNA) and cell survival clearly declined. Recombinant IL-6 treatment weakened the effects of TRPM2-siRNA on cell survival. TRPM2-L binds directly to IL-6R in PC-3 and DU-145 cells. The protein expression of hypoxia-inducible factor-1α was suppressed by reduction with TRPM2-L in PC-3 and DU-145 cells. Human umbilical vein endothelial cells (HUVECs) were indirectly cocultured with PCa cells, and the invasion and angiogenic activity of HUVECs were enhanced after coculture with PCa cells. However, TRPM2-L reduction in PCa cells significantly decreased the invasion and angiogenic activity of HUVECs compared to the control coculture. In vivo, xenograft tumors were induced using PC-3 cells. Tocilizumab treatment or TRPM2-L reduction clearly suppressed tumor growth. Meanwhile, the injection of mouse recombinant IL-6 weakened the antitumor effects of TRPM2-L reduction. These data demonstrate that the IL-6/TRPM2-L axis in PCa tumor growth is important, and interference of the IL-6/TRPM2-L axis may be a novel approach for PCa therapy.

Oral Administration of Universal Bacterium-Vectored Nucleocapsid-Expressing COVID-19 Vaccine is Efficacious in Hamsters.

Oral delivery of an inexpensive COVID-19 (coronavirus disease 2019) vaccine could dramatically improve immunization rates, especially in low- and middle-income countries. Previously, we described a potential universal COVID-19 vaccine, rLVS ΔcapB/MN, comprising a replicating bacterial vector, LVS (live vaccine strain) ΔcapB, expressing the highly conserved SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) membrane and nucleocapsid (N) proteins, which, when administered intradermally or intranasally, protects hamsters from severe COVID-19-like disease after high-dose SARS-CoV-2 respiratory challenge. Here, we show that oral administration of the vaccine also protects against high-dose SARS-CoV-2 respiratory challenge; its protection is comparable to that of intradermal, intranasal, or subcutaneous administration. Hamsters were protected against severe weight loss and lung pathology and had reduced oropharyngeal and lung virus titers. Protection against weight loss and histopathology by the vaccine, which in mice induces splenic and lung cell interferon gamma in response to N protein stimulation, was correlated in hamsters with pre-challenge serum anti-N TH1-biased IgG (IgG2/3). Thus, rLVS ΔcapB/MN has potential as an oral universal COVID-19 vaccine. IMPORTANCE The COVID-19 pandemic continues to rage into its fourth year worldwide. To protect the world's population most effectively from severe disease, hospitalization, and death, a vaccine is needed that is resistant to rapidly emerging viral variants of the causative agent SARS-CoV-2, inexpensive to manufacture, store, and transport, and easy to administer. Ideally, such a vaccine would be capable of oral administration, especially in resource-poor countries of the world where there are shortages of needles, syringes and trained personnel to administer injectable vaccines. Here, we show that oral administration of a bacterium-vectored vaccine meeting all these criteria protects naturally susceptible Syrian hamsters from severe COVID-19-like disease, including severe weight loss and lung pathology, after high-dose SARS-CoV-2 respiratory challenge. As the vaccine is based upon inducing immunity to highly conserved SARS-CoV-2 membrane and nucleocapsid proteins, as opposed to the rapidly mutating Spike protein, it should remain resistant to newly emerging SARS-CoV-2 variants.

Listeria-vectored multi-antigenic tuberculosis vaccine protects C57BL/6 and BALB/c mice and guinea pigs against Mycobacterium tuberculosis challenge.

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.

Listeria-Vectored Multiantigenic Tuberculosis Vaccine Enhances Protective Immunity against Aerosol Challenge with Virulent Mycobacterium tuberculosis in BCG-Immunized C57BL/6 and BALB/c Mice.

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.

Effects of intrastriatal injection of the dopamine receptor agonist SKF38393 and quinpirole on locomotor behavior in hemiparkinsonism rats.

Dopamine (DA) in the striatum is essential to influence motor behavior and may lead to movement impairment in Parkinson's disease (PD). The present study examined the different functions of the DA D1 receptor (D1R) and DA D2 receptor (D2R) by intrastriatal injection of the D1R agonist SKF38393 and the D2R agonist quinpirole in 6-hydroxydopamine (6-OHDA)-lesioned and control rats. All rats separately underwent dose-response behavior testing for SKF38393 (0, 0.5, 1.0, and 1.5 µg/site) or quinpirole (0, 1.0, 2.0, and 3.0 µg/site) to determine the effects of the optimal modulating threshold dose. Two behavior assessment indices, the time of latency to fall and the number of steps on a rotating treadmill, were used as reliable readouts of motor stimulation variables for quantifying the motor effects of the drugs. The findings indicate that at threshold doses, SKF38393 (1.0 µg/site) and quinpirole (1.0 µg/site) produce a dose-dependent increase in locomotor activity compared to vehicle injection. The ameliorated behavioral responses to either SKF38393 or quinpirole in lesioned rats were greater than those in unlesioned control rats. Moreover, the dose-dependent increase in locomotor capacity for quinpirole was greater than that for SKF38393 in lesioned rats. These results can clarify several key issues related to DA receptors directly and may provide a basis for exploring the potential of future selective dopamine therapies for PD in humans.

Roles of Motor Cortex Neuron Classes in Reach-Related Modulation for Hemiparkinsonian Rats.

Disruption of the function of the primary motor cortex (M1) is thought to play a critical role in motor dysfunction in Parkinson's disease (PD). Detailed information regarding the specific aspects of M1 circuits that become abnormal is lacking. We recorded single units and local field potentials (LFPs) of M1 neurons in unilateral 6-hydroxydopamine (6-OHDA) lesion rats and control rats to assess the impact of dopamine (DA) cell loss during rest and a forelimb reaching task. Our results indicated that M1 neurons can be classified into two groups (putative pyramidal neurons and putative interneurons) and that 6-OHDA could modify the activity of different M1 subpopulations to a large extent. Reduced activation of putative pyramidal neurons during inattentive rest and reaching was observed. In addition, 6-OHDA intoxication was associated with an increase in certain LFP frequencies, especially those in the beta range (broadly defined here as any frequency between 12 and 35 Hz), which become pathologically exaggerated throughout cortico-basal ganglia circuits after dopamine depletion. Furthermore, assessment of different spike-LFP coupling parameters revealed that the putative pyramidal neurons were particularly prone to being phase-locked to ongoing cortical oscillations at 12-35 Hz during reaching. Conversely, putative interneurons were neither hypoactive nor synchronized to ongoing cortical oscillations. These data collectively demonstrate a neuron type-selective alteration in the M1 in hemiparkinsonian rats. These alterations hamper the ability of the M1 to contribute to motor conduction and are likely some of the main contributors to motor impairments in PD.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19-like disease in hamsters.

To generate an inexpensive readily manufactured COVID-19 vaccine, we employed the LVS ΔcapB vector platform, previously used to generate potent candidate vaccines against Select Agent diseases tularemia, anthrax, plague, and melioidosis. Vaccines expressing SARS-CoV-2 structural proteins are constructed using the LVS ΔcapB vector, a highly attenuated replicating intracellular bacterium, and evaluated for efficacy in golden Syrian hamsters, which develop severe COVID-19-like disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane and Nucleocapsid proteins and challenged 5 weeks later with a high dose of SARS-CoV-2 are protected against severe weight loss and lung pathology and show reduced viral loads in the oropharynx and lungs. Protection correlates with anti-Nucleocapsid antibody. This potent vaccine should be safe; inexpensive; easily manufactured, stored, and distributed; and given the high homology between Membrane and Nucleocapsid proteins of SARS-CoV and SARS-CoV-2, potentially serve as a universal vaccine against the SARS subset of pandemic causing ß-coronaviruses.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters.

An inexpensive readily manufactured COVID-19 vaccine that protects against severe disease is needed to combat the pandemic. We have employed the LVS Δ capB vector platform, previously used successfully to generate potent vaccines against the Select Agents of tularemia, anthrax, plague, and melioidosis, to generate a COVID-19 vaccine. The LVS Δ capB vector, a replicating intracellular bacterium, is a highly attenuated derivative of a tularemia vaccine (LVS) previously administered to millions of people. We generated vaccines expressing SARS-CoV-2 structural proteins and evaluated them for efficacy in the golden Syrian hamster, which develops severe COVID-19 disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane (M) and Nucleocapsid (N) proteins, then challenged 5-weeks later with a high dose of SARS-CoV-2, were protected against severe weight loss and lung pathology and had reduced viral loads in the oropharynx and lungs. Protection by the vaccine, which induces murine N-specific interferon-gamma secreting T cells, was highly correlated with pre-challenge serum anti-N TH1-biased IgG. This potent vaccine against severe COVID-19 should be safe and easily manufactured, stored, and distributed, and given the high homology between MN proteins of SARS-CoV and SARS-CoV-2, has potential as a universal vaccine against the SARS subset of pandemic causing ß-coronaviruses.

State-Dependent Spike and Local Field Synchronization between the Thalamic Parafascicular Nucleus and the Dorsal Striatum in a Rat Model of Parkinson's Disease.

Recent studies on the impact of Parkinson's disease (PD) on the thalamostriatal pathway have mainly focused on the structural and functional changes in the thalamus projection to the striatum. Alterations in the electrophysiological activity of the thalamostriatal circuit in PD have not been intensively studied. To further investigate this circuit, parafascicular nucleus (PF) single-unit spikes and dorsal striatum local field potential (LFP) activities were simultaneously recorded in control and 6-hydroxydopamine (6-OHDA)-lesioned rats during inattentive rest or treadmill walking states. We classified the PF neurons into two predominant subtypes (PF I and PF II). During rest state, after dopamine loss, increased PF I spike and striatal LFP coherence was observed in the beta-frequency (12-35 Hz), with changed PF I neuronal firing pattern and unchanged firing rates of the two neuron subtypes. However, in a treadmill walking state, PF II neurons displayed markedly increased coherence to striatal beta oscillations in the dopamine-depleted rats, as well as an altered PF II neuronal firing pattern and significantly decreased firing rates of the two neuron subtypes. The results indicate that in PD animals, state transition from rest to moving, such as treadmill walking, is associated with different PF neuron types and increased spike-LFP synchronization, which may provide new paradigms for understanding and treating PD.

Live Attenuated Tularemia Vaccines for Protection Against Respiratory Challenge With Virulent F. tularensis subsp. tularensis.

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.

Single vector platform vaccine protects against lethal respiratory challenge with Tier 1 select agents of anthrax, plague, and tularemia.

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.

Listeria-Vectored Vaccine Expressing the Mycobacterium tuberculosis 30-Kilodalton Major Secretory Protein via the Constitutively Active prfA* Regulon Boosts Mycobacterium bovis BCG Efficacy against Tuberculosis.

A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective Mycobacterium bovis BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein (r30/antigen 85B [Ag85B]) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated L. monocytogenes vectors, L. monocytogenes ΔactA (LmI), L. monocytogenes ΔactA ΔinlB (LmII), and L. monocytogenes ΔactA ΔinlB prfA* (LmIII), we constructed five rLm30 vaccine candidates expressing r30 linked in frame to the L. monocytogenes listeriolysin O signal sequence and driven by the hly promoter (h30) or linked in frame to the ActA N-terminal 100 amino acids and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm30 expressing r30 via a constitutively active prfA* regulon (rLmIII/a30) expressed the largest amount of r30 in broth culture, all five rLm30 vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting of BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T cells expressing the three cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) (P < 0.001) and splenic and lung CD8+ T cells expressing IFN-γ (P < 0.0001). In mice and guinea pigs, the rLmIII/a30 and rLmI/h30 vaccines were generally more potent booster vaccines than r30 with an adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting of mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized M. tuberculosis (P < 0.01).

Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA, IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge.

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.

A heterologous prime-boost vaccination strategy comprising the Francisella tularensis live vaccine strain capB mutant and recombinant attenuated Listeria monocytogenes expressing F. tularensis IglC induces potent protective immunity in mice against virulent F. tularensis aerosol challenge.

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.

A Francisella tularensis live vaccine strain (LVS) mutant with a deletion in capB, encoding a putative capsular biosynthesis protein, is significantly more attenuated than LVS yet induces potent protective immunity in mice against F. tularensis challenge.

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.

Induction of protective immunity against murine gammaherpesvirus 68 infection in the absence of viral latency.

Human gammaherpesviruses, Epstein-Barr virus, and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus are important pathogens associated with diseases, including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) is used as an experimental model system to study the host immune control of infection and explore novel vaccine strategies based on latency-deficient live viruses. We studied the properties and the potential of a recombinant MHV-68 (AC-RTA) in which the genes required for persistent infection were replaced by a constitutively expressed viral transcription activator, RTA, which dictates the virus to lytic replication. After intranasal infection of mice, replication of AC-RTA in the lung was attenuated, and no AC-RTA virus or viral DNA was detected in the isolated splenocytes, indicating a lack of latency in the spleen. Infection of the AC-RTA virus elicited both cellular immune responses and virus-specific IgG at a level comparable to that elicited by infection of the wild-type virus. Importantly, vaccination of AC-RTA was able to protect mice against subsequent challenge by the wild-type MHV-68. AC-RTA provides a vaccine strategy for preventing infection of human gammaherpesviruses. Furthermore, our results suggest that immunity to the major latent antigens is not required for protection.

Acupuncture in the treatment of diabetic bladder dysfunction.

OBJECTIVE: The objective of this study was to investigate the effects of acupuncture on diabetic bladder dysfunction (DBD). METHODS: This study compared 30 cases in the acupuncture group with 15 cases in the sham acupuncture group (n = 45 total). The effects of acupuncture were observed on urodynamic measurements, as well as a variety of symptoms associated with DBD. RESULTS: In the acupuncture group, five of the six urodynamic measures (maximal detrusor pressure, bladder compliance, maximal bladder capacity, bladder volume at desire to void and urge to void) demonstrated significant improvement (p < 0.05, 0.01) over the 15-day treatment period. Only one measure (bladder volume at urge to void) significantly improved (p < 0.05) in the sham acupuncture group. There were significant differences after therapy in four measures (bladder compliance, maximal bladder capacity, bladder volume at desire to void, and urge to void) between the groups (p < 0.05, 0.01). A significant difference of the changes in symptoms compared with pretreatment in the acupuncture group was observed (p < 0.05, 0.01). In 25 subjects in the acupuncture group, incontinence improved from 2.4 to 1.4. In the sham acupuncture group, incontinence deteriorated from 2.2 to 2.3. CONCLUSIONS: Our pilot study has provided evidence that acupuncture may be clinically useful for the radical treatment of DBD.

A renal tuberculosis case: could Chinese medicine play a role?

CASE REPORT: A case of renal tuberculosis (TB) was treated with a multidrug therapeutic regimen (rifampicin 600 mg/day, ethambutol 800 mg/day, and isoniazid 150 mg/day), which was terminated for severe hepatotoxicity 2 months later. As an alternative therapeutic method, the patient was orally administered a Chinese herbal concoction while liver transaminases resumed normal levels. RESULTS: After 1 year of treatment, the patient recovered completely; pyuria and hematuria disappeared with negative acid-fast bacteria urine culture. The patient has been followed up for 2 years without recurrence. CONCLUSIONS: The case indicated that these Chinese herbs are useful in treating renal TB. Chinese medicine has allowed us another choice of antituberculous treatment, avoiding the hepatotoxicity of the standard therapeutic regimen. Therefore, the use of Chinese herbs has the potential of reducing the morbidity and mortality rate of this disease.