Priming Challenge Research Articles

Priming grapevine with lipopolysaccharide confers systemic resistance to Pierce's disease and identifies a peroxidase linked to defense priming.

Priming is an adaptive mechanism that fortifies plant defense by enhancing activation of induced defense responses following pathogen challenge. Microorganisms have signature microbe-associated molecular patterns (MAMPs) that induce the primed state. The lipopolysaccharide (LPS) MAMP isolated from the xylem-limited pathogenic bacterium, Xylella fastidiosa, acts as a priming stimulus in Vitis vinifera grapevines. Grapevines primed with LPS developed significantly less internal tyloses and external disease symptoms than naive vines. Differential gene expression analysis indicated major transcriptomic reprogramming during the priming and postpathogen challenge phases. Furthermore, the number of differentially expressed genes increased temporally and spatially in primed vines, but not in naive vines during the postpathogen challenge phase. Using a weighted gene co-expression analysis, we determined that primed vines have more genes that are co-expressed in both local and systemic petioles than naive vines indicating an inherent synchronicity that underlies the systemic response to this vascular pathogen specific to primed plants. We identified a cationic peroxidase, VviCP1, that was upregulated during the priming and postpathogen challenge phases in an LPS-dependent manner. Transgenic expression of VviCP1 conferred significant disease resistance, thus, demonstrating that grapevine is a robust model for mining and expressing genes linked to defense priming and disease resistance.

Characterization, functional validation, and analysis of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection

Abstract The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 6 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors over multiple infections may create a narrowed repertoire consisting of a few clonotypes, covering a narrow range of antigenic space representing the mutant epitopes encountered. Our data shows that cross-reactive TCRs are more similar to each other than single epitope specific cells and that cross-reactivity can be replicated during peptide stimulation when cross-reactive TCRs are transduced into TCR null cell lines invitro. Given the narrowing nature of cross-reactive responses elicited over different infections, selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools. Supported by NIH Grant R01112404040 and the St. Jude Graduate School of Biomedical Sciences

Characterization of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection

Abstract The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. The number of possible peptides presented to the immune system exceeds 1015, and this number is greater than the number of possible T cells the immune system can generate and sustain. Thus, a certain portion of the T cells in an immune system must be cross-reactive. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 8 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors may create a narrowed repertoire consisting of a few clonotypes over the course of many repeated heterosubtypic IAV infections, covering a narrow range of antigenic space representing the mutant epitopes encountered. Paradoxically, this selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools. Supported by grants from NIH (R01 112404040) and the St. Jude Children's Research Hospital Graduate School

Generalized Shwartzman Reaction as an Experimental Endotoxin Shock Model: Role of Intermediate T Cell Receptor-Expressing Innate T Lymphocytes in its Pathogenesis

Endotoxin and its induced exaggerative responses of proinflammatory cytokines such as Tumor Necrosis Factor (TNF) cause human septic shock. The generalized Shwartzman reaction is considered as an experimental endotoxin shock model and its representative features are high lethality and hypersensitive inflammatory response to endotoxin, in particular excessive TNF production. The generalized Shwartzman reaction can be induced by Interleukin (IL)-12 priming and subsequent endotoxin challenge. IL-12 primes the intermediate T cell receptor (TCR)-expressing innate T lymphocytes (natural killer T cells and CD8+CD122+ T cells in mice, CD56+ T cells and CD57+ T cells in human) to produce IFN-γ, which in turn preconditions macrophages to produce a large amount of TNF in response to endotoxin. These intermediate TCR innate T lymphocytes play a pivotal role for the occurrence of this generalized Shwartzman reaction. Some of the intermediate TCR-expressing innate T lymphocytes (CD8+CD122+ T cells in mice and CD57+ T cells in human), which are considered as extrathymically developed T lymphocytes, are increased with age and closely involved in age-dependent augmentation of murine generalized Shwartzman reaction and human Shwartzman-like reaction, which may reflect an exacerbation of septic shock in elderly. However, these intermediate TCR innate T lymphocytes normally augment host immunity against malignant tumors and refractory bacterial infections, which are critical threats in aged hosts. Thus, intermediate TCR-expressing innate T lymphocytes may be a double-edged sword on the host defense, especially elderly hosts.

Characterization of the cross reactive T cell repertoire in the context of heterosubtypic influenza A virus infection

Abstract Influenza A viruses (IAVs) are a major respiratory pathogen with the capacity to rapidly mutate and escape an individual’s existing immunity to a specific strain generated by vaccination or naturally acquired infection. CD8 T cells however, target conserved proteins that could be shared across multiple subtypes. Heterosubtypic immunity to IAVs, where memory T cells generated to a previous IAV are reactivated during re-challenge with an antigenically distinct virus, may be important in generating an effective immune response to different and/or novel strains of IAV. Specifically, cross reactive T cells, where a T cell receptor (TCR) can recognize more than one epitope displayed on an MHC-I molecule, could facilitate rapid recognition of conserved or nominally divergent epitopes, may have the potential to alter the TCR repertoire over the course of heterosubtypic infections, and might be selected for by the immune system. Utilizing 8 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, even if the epitope has slightly mutated. However, selection for cross reactive receptors may create a narrowed repertoire consisting of a few clonotypes over the course of many repeated heterosubtypic IAV infections, covering a narrow range of antigenic space representing the mutant epitopes encountered. Paradoxically, this selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape and are shaped by these selected cross-reactive T cell pools.

Determining Immune and miRNA Biomarkers Related to Respiratory Syncytial Virus (RSV) Vaccine Types.

Respiratory Syncytial Virus (RSV) causes serious respiratory tract illness and substantial morbidity and some mortality in populations at the extremes of age, i.e., infants, young children, and the elderly. To date, RSV vaccine development has been unsuccessful, a feature linked to the lack of biomarkers available to assess the safety and efficacy of RSV vaccine candidates. We examined microRNAs (miR) as potential biomarkers for different types of RSV vaccine candidates. In this study, mice were vaccinated with a live attenuated RSV candidate that lacks the small hydrophobic (SH) and attachment (G) proteins (CP52), an RSV G protein microparticle (GA2-MP) vaccine, a formalin-inactivated RSV (FI-RSV) vaccine or were mock-treated. Several immunological endpoints and miR expression profiles were determined in mouse serum and bronchoalveolar lavage (BAL) following vaccine priming, boost, and RSV challenge. We identified miRs that were linked with immunological parameters of disease and protection. We show that miRs are potential biomarkers providing valuable insights for vaccine development.

The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

The NLRP3 inflammasome has an important function in inflammation by promoting the processing of pro-IL-1β and pro-IL-18 to their mature bioactive forms, and by inducing cell death via pyroptosis. Here we show a critical function of the E3 ubiquitin ligase Pellino2 in facilitating activation of the NLRP3 inflammasome. Pellino2-deficient mice and myeloid cells have impaired activation of NLRP3 in response to toll-like receptor priming, NLRP3 stimuli and bacterial challenge. These functions of Pellino2 in the NLRP3 pathway are dependent on Pellino2 FHA and RING-like domains, with Pellino2 promoting the ubiquitination of NLRP3 during the priming phase of activation. We also identify a negative function of IRAK1 in the NLRP3 inflammasome, and describe a counter-regulatory relationship between IRAK1 and Pellino2. Our findings reveal a Pellino2-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome.

Music therapy inhibits morphine-seeking behavior via GABA receptor and attenuates anxiety-like behavior induced by extinction from chronic morphine use

RationaleMorphine is a representative pain killer. However, repeated use tends to induce addiction. Music therapy has been gaining interest as a useful type of therapy for neuropsychiatric diseases. ObjectivesThe present study examined whether Korean traditional music (KT) could suppress morphine-seeking behavior and anxiety-like behavior induced by extinction from chronic morphine use and additionally investigated a possible neuronal mechanism. Material & methodsMale Sprague-Dawley rats were trained to intravenously self-administer morphine hydrochloride (1.0 mg/kg) using a fixed ratio 1 schedule in daily 2 h session during 3 weeks. After training, rats who established baseline (variation less than 20% of the mean of infusion for 3 consecutive days) underwent extinction. Music was played twice a day during extinction. In the second experiment, the selective antagonists of GABAA and GABAB receptors were treated before the last playing to investigate the neuronal mechanism focusing on the GABA receptor pathway. Another experiment of elevated plus maze was performed to investigate whether music therapy has an anxiolytic effect at the extinction phase. ResultsKT but not other music (Indian road or rock music) reduced morphine-seeking behavior induced by a priming challenge with morphine. And, this effect was blocked by the GABA receptor antagonists. In addition, KT showed anxiolytic effects against withdrawal from morphine. ConclusionsResults of this study suggest that KT suppresses morphine-seeking behavior via GABA receptor pathway. In addition, KT showed to have anxiolytic effects, suggesting it has bi-directional effects on morphine.

Oral immune priming with Bacillus thuringiensis induces a shift in the gene expression of Tribolium castaneum larvae

BackgroundThe phenomenon of immune priming, i.e. enhanced protection following a secondary exposure to a pathogen, has now been demonstrated in a wide range of invertebrate species. Despite accumulating phenotypic evidence, knowledge of its mechanistic underpinnings is currently very limited. Here we used the system of the red flour beetle, Tribolium castaneum and the insect pathogen Bacillus thuringiensis (Bt) to further our molecular understanding of the oral immune priming phenomenon. We addressed how ingestion of bacterial cues (derived from spore supernatants) of an orally pathogenic and non-pathogenic Bt strain affects gene expression upon later challenge exposure, using a whole-transcriptome sequencing approach.ResultsWhereas gene expression of individuals primed with the orally non-pathogenic strain showed minor changes to controls, we found that priming with the pathogenic strain induced regulation of a large set of distinct genes, many of which are known immune candidates. Intriguingly, the immune repertoire activated upon priming and subsequent challenge qualitatively differed from the one mounted upon infection with Bt without previous priming. Moreover, a large subset of priming-specific genes showed an inverse regulation compared to their regulation upon challenge only.ConclusionsOur data demonstrate that gene expression upon infection is strongly affected by previous immune priming. We hypothesise that this shift in gene expression indicates activation of a more targeted and efficient response towards a previously encountered pathogen, in anticipation of potential secondary encounter.

Response to Ragweed Allergen Provocation in the Red Maple Trials Allergen Challenge Theatre

Allergen challenge chambers expose allergen-sensitive subjects to predetermined concentrations of allergen in a controlled environment and provide a mechanism to induce clinical symptoms and measure the effect of medication. We evaluated the response of ragweed-allergic subjects to two ragweed challenges in the Red Maple Trials Allergen Challenge Theatre. A provincial Ethics Board approved the study. After signing informed consent, patients with a history of ragweed allergy, not on allergy medications and with positive skin prick tests (> 3 mm) to ragweed antigen were exposed to ragweed pollen in a 3-hour priming session. Total nasal (TNSS), ocular and rhinoconjunctivitis symptom scores (TRSS) were recorded at baseline and every 30 minutes during the challenge. Those with peak TNSS ≥ 5 were then selected for two further 4-hour challenges. 48/76 subjects evaluated underwent the priming challenge. Thirty-three subjects achieving a peak TNSS ≥5 underwent two subsequent 4-hour challenges. Baseline TNSS (mean± SD) was 1.70 ±1.34 and 2.53±1.76 in challenges 1 and 2, respectively. Baseline TRSS values were 2.00± 1.66 and 3.60±2.69 respectively. Symptom scores reached a plateau by 120 minutes and remained steady for the remainder of the 240-minute exposure. Plateau TNSS was 6.28±0.20 for Challenge 1 and 6.19±0.24 for Challenge 2. Similarly, plateau TRSS values were 9.10±0.20 and 9.11± 0.33, respectively. The Red Maple Trials allergen challenge theatre demonstrated the capacity to induce symptoms of appropriate intensity upon ragweed allergen challenge. The chamber with a 100-person capacity has the ability to evaluate large test groups at one time.

Changes in Explanatory Flexibility Among Individuals with Generalized Anxiety Disorder in an Emotion Evocation Challenge

Seventy-eight undergraduates, 39 with self-reported generalized anxiety disorder (GAD), completed measures of mood and explanatory flexibility (the capacity to assign causes to negative events with a balance of historical and contextual factors) prior to and directly after a musical priming challenge that consisted of listening to negatively-valenced emotional music and thinking about a personally relevant negative event. After the emotion evocation, participants also completed a measure of state emotion regulation. Despite comparable increases in negative affect, GAD analogues evidenced drops in explanatory flexibility whereas non-GAD Controls did not. Drops in explanatory flexibility among GAD analogues covaried significantly with lack of emotional clarity. Findings suggest that for individuals with GAD, emotionally evocative experiences may result in a constricted perspective when apprehending the causes for negative events. This perspective may serve to dampen arousal, but perhaps at the cost of failing to inform one’s actions with important emotional information.

Characterizing low dose and dose rate effects in rodent and human neural stem cells exposed to proton and gamma irradiation

Past work has shown that exposure to gamma rays and protons elicit a persistent oxidative stress in rodent and human neural stem cells (hNSCs). We have now adapted these studies to more realistic exposure scenarios in space, using lower doses and dose rates of these radiation modalities, to further elucidate the role of radiation-induced oxidative stress in these cells. Rodent neural stem and precursor cells grown as neurospheres and human neural stem cells grown as monolayers were subjected to acute and multi-dosing paradigms at differing dose rates and analyzed for changes in reactive oxygen species (ROS), reactive nitrogen species (RNS), nitric oxide and superoxide for 2 days after irradiation. While acute exposures led to significant changes in both cell types, hNSCs in particular, exhibited marked and significant elevations in radiation-induced oxidative stress. Elevated oxidative stress was more significant in hNSCs as opposed to their rodent counterparts, and hNSCs were significantly more sensitive to low dose exposures in terms of survival. Combinations of protons and γ-rays delivered as lower priming or higher challenge doses elicited radioadaptive changes that were associated with improved survival, but in general, only under conditions where the levels of reactive species were suppressed compared to cells irradiated acutely. Protective radioadaptive effects on survival were eliminated in the presence of the antioxidant N-acetylcysteine, suggesting further that radiation-induced oxidative stress could activate pro-survival signaling pathways that were sensitive to redox state. Data corroborates much of our past work and shows that low dose and dose rate exposures elicit significant changes in oxidative stress that have functional consequences on survival.

Long-lasting transcriptional refractoriness triggered by a single exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine

Parkinson’s disease (PD) is a progressive neurodegenerative disorder whose etiology is thought to have environmental (toxin) and genetic contributions. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP) induces pathological features of PD including loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and striatal dopamine (DA) depletion. We previously described the striatal transcriptional response following acute MPTP administration in MPTP-sensitive C57BL/6J mice. We identified three distinct phases: early (5h), intermediate (24h) and late (72h) and reported that the intermediate and late responses were absent in MPTP-resistant Swiss-Webster (SWR) mice. Here we show that C57BL/6J mice pre-treated with a single 40mg/kg dose of MPTP and treated 9days later with 4×20mg/kg MPTP, display a striatal transcriptional response similar to that of MPTP-resistant SWR mice, i.e. a robust acute response but no intermediate or late response. Transcriptional refractoriness is dependent upon the dose of the priming challenge with as little as 10mg/kg MPTP being effective and can persist for more than 28days. Priming of SWR mice has no effect on their response to subsequent challenge with MPTP. We also report that paraquat, another free radical producer, also elicits striatal transcriptional alterations but these are largely distinct from those triggered by MPTP. Paraquat-induced changes are also refractory to priming with paraquat. However neither paraquat nor MPTP elicits cross-attenuation. Thus exposure to specific toxins triggers distinct transcriptional responses in striatum that are influenced by prior exposure to the same toxin. The prolonged refractory period described here for MPTP could explain at the molecular level the reported discrepancies between different MPTP administration regimens and may have implications for our understanding of the relationship between environmental toxin exposure and PD.

The effect of a negative mood priming challenge on dysfunctional attitudes, explanatory style, and explanatory flexibility

Ninety-seven undergraduates, 48 of whom had a history of self-reported major depression, completed measures of mood and cognitive style (e.g. explanatory style, explanatory flexibility, dysfunctional attitudes) prior to and directly after a negative mood priming challenge that consisted of listening to sad music and thinking about an upsetting past event. Eighteen of the previously depressed participants endorsed baseline levels of depression, explanatory style for negative events, and dysfunctional attitudes higher than levels reported by never depressed participants or euthymic participants with a history of depression. All three groups (never depressed participants, dysphoric participants with a history of depression, euthymic participants with a history of depression) demonstrated increases in dysphoria and dysfunctional attitudes in response to the negative mood priming challenge. Dysphoric participants with a history of depression, but not the other two groups, evidenced modest increases in explanatory style following the negative mood priming challenge. Finally, euthymic participants with a history of depression, but not the other two groups, evidenced drops in explanatory flexibility. Findings from the present study suggest that the cognitive theories of depression may benefit from examining both cognitive content and cognitive flexibility when assessing risk for depression.

Critical role of the liver CD8+ CD122+ T cells in the generalized Shwartzman reaction of mice

We examined the role of mouse CD8+ CD122+ T cells, which increase in number with age, in the generalized Shwartzman reaction. This reaction was induced by IL-12 priming and subsequent LPS challenge (after 24 h) in mice of various ages (4-50 weeks of age). Although most young mice (4 or 6 weeks of age) survived, mortality essentially increased with increasing age of the mice, and all mice of 20 weeks of age or older died within 48 h. Serum TNF-alpha levels after LPS challenge also increased age dependently. The neutralization of either IL-12-induced IFN-gamma or LPS-induced TNF-alpha improved the survival of middle-aged (25-week-old) mice. Both IFN-gamma production after IL-12 priming and TNF-alpha production from the liver mononuclear cells after LPS challenge were also prominent in the middle-aged mice. CD8+CD122+ T cells cultured with IL-12 produced a much larger amount of IFN-gamma than CD8+CD122- T cells. Although the depletion of NK/NK T cells did not decrease the IFN-gamma or TNF-alpha production in the Shwartzman reaction of the middle-aged mice, an additional depletion of CD8+CD122+ T cells did decrease such production and also improved mouse survival. Furthermore, young mice transferred with CD8+CD122+ T cells from aged B6 nude mice showed an enhanced Shwartzman reaction.

Roles of CD14 in LPS-induced liver injury and lethality in mice pretreated with Propionibacterium acnes

The mechanism of the liver damage and lethaliy in Propionibacterium acnes ( P. acnes)-LPS system remains obscure. To examine the role of CD14 in the system, M14M mice, in which CD14 was expressed heterotopically under the control of the metallothionein promoter were used. The production of soluble CD14 (sCD14) was increased by both P. acnes – priming and LPS challenge (1 μg per mouse) in both nontransgenic and M14M mice, although the plasma level was much higher in M14M nontransgenic than mice. The size of granulomas induced by an intraperitoneal administration of P. acnes in M14M mice 7 days after priming was smaller than that in nontransgenic mice. An LPS challenge induced apoptotic and necrotic changes in hepatocytes in nontransgenic mice but not in M14M mice. The challenge dose resulted in almost 90% lethality in nontransgenic mice but not in M14M mice 24 h after challenge. TNF-α, IFN-γ, IL-12, IL-18 and inducible nitric oxide synthase (iNOS) mRNA expressions produced by LPS challenge in M14M mice were low compared with those in nontransgenic mice. IL-18 mRNA expression was upregulated in P. acnes-primed nontransgenic mice but not in M14M mice. These results suggest that the high sCD14 concentration may account for less marked liver damage in M14M mice. Increase in the challenge dose of LPS (2 μg per mouse) resulted in increased lethality of M14M mice without liver damage. The levels of endothelial cell leukocyte adhesion molecule (ELAM)-1 mRNA expression in several organs in M14M mice 1–3 h after LPS challenge were, however, lower than those in nontransgenic mice. The high sCD14 concentration may stimulate endothelial cell activation, which may account for lethality without liver damage in M14M mice. Thus, CD14 is involved in both the priming and induction phases as well as lethality in P. acnes-LPS system.

Involvement of CD14 in LPS-induced liver injury in Propionibacterium acnes-primed mice

The size of granulomas induced by an intraperitoneal administration of Propionibacterium acnes in M14M mice 7 days after priming was smaller than that in non-transgenic mice. The number of CD14-positive cells in granulomas was fewer in M14M mice than in non-transgenic mice. An LPS challenge induced apoptotic and necrotic changes in hepatocytes in non-transgenic mice but not in M14M mice. TNFα expression was found in monocytic cells in granulomas and Kupffer cells in non-transgenic mice 7 days after priming and was significantly upregulated after LPS injection, whereas the expression was very low in these cells in M14M mice. The expression of IFN-γ, IL-12 and iNOS mRNAs produced by LPS challenge in M14M mice were low compared with those in non-transgenic mice. IL-18 mRNA expression was upregulated in P. acnes-primed nontransgenic mice but not in M14M mice. The production of sCD14 was increased by both P. acnes priming and LPS challenge. The high sCD14 concentration may account for lethality without liver damage in M14M mice. The levels of ELAM-1 mRNA expression in several organs in M14M mice 1—3 h after LPS challenge were, however, lower than those in non-transgenic mice.

Involvement of NK1+ T Cells and Their IFN-γ Production in the Generalized Shwartzman Reaction

AbstractIL-12 (or LPS) priming and subsequent challenge by LPS produces the generalized Shwartzman reaction. IFN-γ induced by IL-12 is a crucial cytokine in the priming phase. In vivo depletion of both NK cells and NK1+ αβ T cells of mice by anti-NK1.1 Ab greatly reduced the elevation of serum IFN-γ induced by IL-12 and significantly reduced mortality after subsequent injection of LPS, whereas depletion of NK cells alone by anti-asialo GM1 Ab only partially decreased serum IFN-γ, and lethality was not changed. Cell sorting and culture experiments confirmed that liver NK1+ αβ T cells of IL-12-injected mice produced greater amounts of IFN-γ than did liver NK cells. MHC class I-deficient mice of C57BL/6 background, which lack a majority of NK1+ αβ T cells, produced low amounts of IFN-γ by IL-12; no mortality was observed after the LPS challenge. However, production of TNF-α in the second phase (after LPS challenge) was not inhibited by depletion of NK cells alone or both subsets. IL-12 and subsequent LPS challenge activated NK1+ αβ T cells in the liver and induced strong cytotoxicity of these cells not only against tumor cells (including Fas-negative tumors) but also against a syngeneic hepatocyte cell line. Our findings show that IFN-γ produced by NK1+ αβ T cells is essential for the IL-12 priming of the Shwartzman reaction, and the autoreactivity of NK1+ αβ T cells in the liver is involved in the hepatic disorders that are sometimes caused by IL-12, LPS, or the generalized Shwartzman reaction.

Synergism between endotoxin priming and exotoxin challenge in provoking severe vascular leakage in rabbit lungs.

Lipopolysaccharides (LPS) of gram-negative bacteria prime rabbit lungs for enhanced thromboxane-mediated vasoconstriction upon subsequent challenge with the exotoxin Escherichia coli hemolysin (HlyA) (Walmrath et al. J. Exp. Med. 1994;180:1437-1443). We investigated the impact of endotoxin priming and subsequent HlyA challenge on lung vascular permeability while maintaining constancy of capillary pressure. Rabbit lungs were perfused in a pressure-controlled mode in the presence of the thromboxane receptor antagonist BM 13.505, with continuous monitoring of flow. Perfusion for 180 min with 10 ng/ml LPS did not provoke vasoconstriction or alteration of capillary filtration coefficient (Kfc) values. HlyA (0.021 hemolytic units/ml) induced thromboxane release and a transient decrease in perfusion flow in the absence of significant changes in Kfc. Similar results were obtained when LPS and HlyA were coapplied simultaneously. However, when the HlyA challenge was undertaken after 180 min of LPS priming, a manifold increase in Kfc values was noted, with concomitant severe lung edema formation, although capillary pressure remained unchanged. Thus, endotoxin primes the lung vasculature to respond with a severe increase in vascular permeability to a subsequent low-dose application of HlyA. Such synergism between endotoxin priming and exotoxin challenge in provoking lung vascular leakage may contribute to the pathogenesis of respiratory failure in sepsis and severe lung infection.

Augmentation of antimetastatic effect on Lewis lung carcinoma (3LL) in C57BL/6 mice by priming with Lactobacillus casei

The augmentation of the antimetastatic effect of heat-killed cells of Lactobacillus casei YIT9018 (LC9018) on Lewis lung carcinoma (3LL) in C57BL/6 mice by presensitization (priming) with LC9018 was examined. Intralesional injection of LC9018 into 3LL-bearing mice inhibited both the growth of the primary tumors and the formation of lung metastases, and this effect was significantly augmented by subcutaneous injection of LC9018 before the tumor inoculation. In the LC9018-primed mice, intraperitoneal administration of LC9018 into syngeneic hosts after priming induced a high level of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in the peritoneal cavity. At this time, T cells of the spleen cells from the LC9018-primed mice proliferated and produced IL-2 when co-cultured with LC9018 as antigen in vitro. Also, the phenotype of these T cells was found to be L3T4+ and Ly-2.2- T cells by analysis by flow cytometry. These results suggest that LC9018-reactive helper T (Th) cells were induced by the priming and subsequent challenge with LC9018, and that IL-2 or IFN-gamma, which was produced by the activated LC9018-reactive Th cells, augmented a host immune response resulting the antitumor activity.