Systemic cytokine and chemokine responses in immunized mice challenged with staphylococcal enterotoxin B.

The cytokine storm induced by staphylococcal enterotoxin B (SEB) describes the rapid and dramatic induction of mediators which are likely responsible for the toxin's deleterious effects. However despite the use of numerous animal models for investigating SEB related illness in humans, mechanisms of toxicity and correlates of protection remain unclear. In the present study, we used an LPS-potentiated model of SEB lethality to investigate the toxin-induced cytokine and chemokine responses in untreated and immunized mice. Of 30 separate mediators analyzed, serum levels for 28 or 27 of these cytokines and chemokines were elevated following administration of dosages of 3 or 30 LD50 of native SEB, respectively. Mice immunized with a non-toxic SEB vaccine candidate expressed in either E. coli or transgenic soy expression systems were protected from lethality when challenged with potentiated SEB. The majority of SEB-induced cytokines and chemokines (21 of 28 or 23 of 27 following challenge with dosages of 3 or 30 LD50 of native SEB, respectively) were significantly decreased in mice immunized with an SEB vaccine candidate when compared to control animals. Together, these studies provide the most comprehensive evaluation of the cytokine storm induced in this LPS-potentiated model of SEB lethality to date. As with other animal models, the identification of those mediators which are necessary and sufficient for SEB-induced toxicity remains unclear.

Transcript Polymorphism Rates in Soybean Seed Tissue Are Increased in a Single Transformant of Glycine max.

Transgenic crops have been utilized for decades to enhance agriculture and more recently have been applied as bioreactors for manufacturing pharmaceuticals. Recently, we investigated the gene expression profiles of several in-house transgenic soybean events, finding one transformant group to be consistently different from our controls. In the present study, we examined polymorphisms and sequence variations in the exomes of the same transgenic soybean events. We found that the previously dissimilar soybean line also exhibited markedly increased levels of polymorphisms within mRNA transcripts from seed tissue, many of which are classified as gene expression modifiers. The results from this work will direct future investigations to examine novel SNPs controlling traits of great interest for breeding and improving transgenic soybean crops. Further, this study marks the first work to investigate SNP rates in transgenic soybean seed tissues and demonstrates that while transgenesis may induce abundant unanticipated changes in gene expression and nucleotide variation, phenotypes and overall health of the plants examined remained unaltered.

A Sublethal Swine Model for Defining In Vivo Superantigen-Induced Responses Following Exposure to Staphylococcal Enterotoxin B.

In vivo responses to bacterially derived superantigen-like toxins have been difficult to define due to the inherent limitations with rodent models and the relevance that the results obtained from such models may, or may not, have for human pathophysiology. Further the use of challenge doses of superantigen toxins that are lethal or supra-lethal complicates analogies to human exposures which are rarely fatal. Here, we utilize the superantigen, staphylococcal enterotoxin B, at doses that are sublethal in a swine model of toxin-induced incapacitation. Relevant dosing using an animal species for which this toxin is a true superantigen distinguishes this model.

CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing.

Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.

A Comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq.

BACKGROUND: Soybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene. METHODS: Each transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting.  Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing.  Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks.  Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO. RESULTS: The transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected. CONCLUSIONS: Measurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.

Soybean seeds: a practical host for the production of functional subunit vaccines.

Soybean seeds possess several inherent qualities that make them an ideal host for the production of biopharmaceuticals when compared with other plant-based and non-plant-based recombinant expression systems (e.g., low cost of production, high protein to biomass ratio, long-term stability of seed proteins under ambient conditions, etc.). To demonstrate the practicality and feasibility of this platform for the production of subunit vaccines, we chose to express and characterize a nontoxic form of S. aureus enterotoxin B (mSEB) as a model vaccine candidate. We show that soy-mSEB was produced at a high vaccine to biomass ratio and represented ~76 theoretical doses of human vaccine per single soybean seed. We localized the model vaccine candidate both intracellularly and extracellularly and found no difference in mSEB protein stability or accumulation relative to subcellular environment. We also show that the model vaccine was biochemically and immunologically similar to native and recombinant forms of the protein produced in a bacterial expression system. Immunization of mice with seed extracts containing mSEB mounted a significant immune response within 14 days of the first injection. Taken together, our results highlight the practicality of soybean seeds as a potential platform for the production of functional subunit vaccines.

Alpha beta-crystallin expression and presentation following infection with murine gammaherpesvirus 68.

Alpha beta-crystallin (CRYAB) is a small heat shock protein that can function as a molecular chaperone and has protective effects for cells undergoing a variety of stressors. Surprisingly, CRYAB has been identified as one of the dominant autoantigens in multiple sclerosis. It has been suggested that autoimmune mediated destruction of this small heat shock protein may limit its protective effects, thereby exacerbating inflammation and cellular damage during multiple sclerosis. It is not altogether clear how autoimmunity against CRYAB might develop, or whether there are environmental factors which might facilitate the presentation of this autoantigen to CD4+ T lymphocytes. In the present study, we utilized an animal model of an Epstein Barr Virus (EBV)-like infection, murine gammaherpesvirus 68 (HV-68), to question whether such a virus could modulate the expression of CRYAB by antigen presenting cells. Following exposure to HV-68 and several other stimuli, in vitro secretion of CRYAB and subsequent intracellular accumulation were observed in cultured macrophages and dendritic cells. Following infection of mice with this virus, it was possible to track CRYAB expression in the spleen and in antigen presenting cell subpopulations, as well as its secretion into the blood. Mice immunized with human CRYAB mounted a significant immune response against this heat shock protein. Further, dendritic cells that were exposed to HV-68 could stimulate CD4+ T cells from CRYAB immunized mice to secrete interferon gamma. Taken together these studies are consistent with the notion of a gammaherpesvirus-induced CRYAB response in professional antigen presenting cells in this mouse model.

Sublethal staphylococcal enterotoxin B challenge model in pigs to evaluate protection following immunization with a soybean-derived vaccine.

In an effort to develop a sustainable platform for manufacturing protein-based vaccine candidates, we expressed a triple mutant of staphylococcal enterotoxin B carrying the L45R, Y89A, and Y94A modifications in transgenic soybean seeds (soy-mSEB). Soy-mSEB possessed no detectable superantigen activity in vitro. We found that this soybean-derived, nontoxic mutant of SEB could be stably expressed, stored in seeds for extended periods at room temperature without degradation, and easily purified from contaminating soy proteins. Vaccination of pigs with purified soy-mSEB, or the identical triple mutant expressed in Escherichia coli (E. coli-mSEB), resulted in high antibody titers against the native toxin in immunized animals. In fact, titers were indistinguishable regardless of the immunogen used, demonstrating the equivalence of soy-mSEB and E. coli-mSEB vaccinations. Antisera from either immunized group were able to block native SEB superantigen activity in an in vitro neutralization assay. Similar results were obtained when immunized animals were challenged with a sublethal dose of native toxin. Significant reductions in toxin-induced serum cytokine levels were observed in soy-mSEB- and E. coli-mSEB-immunized pigs compared to control animals. The reductions in SEB-induced cytokine responses were similar regardless of the immunogen used for vaccination. Surprisingly, however, some clinical symptoms, such as prostration, lethargy, emesis, and/or diarrhea, were still observed in all immunized animals. These studies demonstrate the potential for soybean-derived proteins as a platform technology for sustainable vaccine manufacturing and the usefulness of a sublethal challenge model in pigs for evaluating the efficacy of potential SEB vaccine candidates.

An expanded myeloid derived suppressor cell population does not play a role in gammaherpesvirus-exacerbated breast cancer metastases.

BACKGROUND: Mice latently infected with murine gammaherpesvirus 68 (HV-68) and transplanted with 4 T1 breast cancer cells developed exacerbated metastatic lesions when compared to controls. The mechanisms responsible for this viral-exacerbated disease were not clear. The ability of HV-68 infection to induce S100A8 and S100A9 production and to expand a population of CD11b+Gr-1+ cells suggested that increased numbers, or activity, of viral-expanded myeloid derived suppressor cells (MDSCs) might contribute to HV-68-associated metastatic breast cancer in this model. We questioned whether mock or HV-68 infected mice with significant breast cancer might have differences in the number and/or activity of MDSCs. METHODS: Myeloid-derived macrophages and dendritic cells were isolated from normal mice and cultured in vitro with HV-68 to assess S100A8 and S100A9 mRNA and protein expression. In vivo studies were performed using groups of mice that were mock treated or infected with HV-68. After viral latency was established, 4 T1 breast cancer cells were transplanted in mice. When primary breast tumors were present mice were euthanized and cells isolated for phenotyping of myeloid cell populations using FACS, and for ex vivo analysis of suppressor activity. Serum from these animals was also collected to quantify S100A8 and S100A9 levels. RESULTS: In vitro studies demonstrated that direct exposure of myeloid cells to HV-68 did not induce increased expression of S100A8 or S100A9 mRNAs or secreted protein. HV-68 infected mice with metastatic breast cancer disease had no increases in S100A8/A9 levels and no significant increases in the numbers or activation of CD11b+Gr-1+MDSCs when compared to mock treated mice with breast cancer. CONCLUSIONS: Together these studies are consistent with the notion that expanded myeloid derived suppressor cells do not play a role in gammaherpesvirus-exacerbated breast cancer metastases. The mechanisms responsible for HV-68 induced exacerbation of metastatic breast cancer remain unclear.

Exacerbated metastatic disease in a mouse mammary tumor model following latent gammaherpesvirus infection.

BACKGROUND: Controversy exists as to the ability of human gammaherpesviruses to cause or exacerbate breast cancer disease in patients. The difficulty in conducting definitive human studies can be overcome by investigating developing breast cancer in a mouse model. In this study, we utilized mice latently infected with murine gammaherpesvirus 68 (HV-68) to question whether such a viral burden could exacerbate metastatic breast cancer disease using a mouse mammary tumor model. RESULTS: Mice latently infected with HV-68 had a similar primary tumor burden, but much greater metastatic disease, when compared to mock treated mice given the transplantable tumor, 4 T1. This was true for lung lesions, as well as secondary tumor masses. Increased expression of pan-cytokeratin and VEGF-A in tumors from HV-68 infected mice was consistent with increased metastatic disease in these animals. Surprisingly, no viral particles could be cultured from tumor tissues, and the presence of viral DNA or RNA transcripts could not be detected in primary or secondary tumor tissues. CONCLUSIONS: Latent HV-68 infection had no significant effect on the size of primary 4 T1 mammary tumors, but exacerbated the number of metastatic lung lesions and secondary tumors when compared to mock treated mice. Increased expression of the tumor marker, pan-cytokeratin, and VEGF-A in tumors of mice harboring latent virus was consistent with an exacerbated metastatic disease. Mechanisms responsible for this exacerbation are indirect, since no virus could be detected in cancerous tissues.

Murine gammaherpesvirus-68 expands, but does not activate, CD11b+ gr-1+ splenocytes in vivo.

BACKGROUND: Murine gammaherpesvirus 68 (HV-68) is an efficient pathogen, capable of infecting and establishing lifelong latency in rodents. While many studies have demonstrated the ability of this viral infection to modulate immune responses, a unifying mechanism for HV-68-induced subversion of a protective host response remains elusive. We questioned whether infection with HV-68 could expand a population of myeloid derived suppressor cells (MDSC) as one mechanism for altering protective immunity. METHODS: Mice were infected with HV-68, with viral latency being established in these animals. At varying times post-infection, cells were isolated for detection of viral genomes, phenotyping of myeloid cell populations, and ex vivo analysis of suppressor activity of myeloid cells. RESULTS: CD11b + Gr-1+ myeloid cells accumulated in the spleens, but not the bone marrow, of HV-68 infected mice. These cells were predominantly Gr-1+ Ly-6 G+, and could be found to contain viral genomes. Increased levels of serum S100A8/A9 produced during viral infection were consistent with the expansion of these CD11b + Gr-1+ myeloid cells. Despite their expansion, these cells exhibited no increased arginase 1 or iNOS activity, and did not have the ability to suppress anti-CD3 antibody activated T lymphocyte responses. CONCLUSIONS: We concluded that HV-68 infection was capable of expanding a population of myeloid cells which were phenotypically similar to MDSC. However these cells were not sufficiently activated during the establishment of viral latency to actively suppress T cell responses.

Prophylactic and therapeutic targeting of the neurokinin-1 receptor limits neuroinflammation in a murine model of pneumococcal meningitis.

There is increasing evidence that the tachykinin substance P (SP) can augment inflammatory immune responses within the CNS. We have recently demonstrated that resident CNS cells express high-affinity receptors for this neuropeptide (neurokinin-1 receptors [NK-1R]), and we have shown that SP can significantly augment glial inflammatory responses to clinically relevant Gram-negative bacteria. Furthermore, we provided evidence that endogenous SP/NK-1R interactions are an essential component in the initiation and/or progression of CNS inflammation following in vivo exposure to these pathogens. In this study, we demonstrate that SP similarly enhances inflammatory glial responses to the major Gram-positive causative agent of bacterial meningitis, Streptococcus pneumoniae, and show that endogenous SP/NK-1R interactions play a critical role in the development of CNS inflammation in an in vivo model of pneumococcal meningitis. Importantly, we provide the first demonstration, to our knowledge, that pharmacological targeting of the NK-1R not only prevents the development of damaging inflammation when administered prophylactically, but can also limit or reverse neuroinflammation associated with an established streptococcal CNS infection when delivered therapeutically. We show that an NK-1R antagonist attenuates increases in CNS inflammatory cytokine levels and decreases in immunosuppressive cytokine production associated with an ongoing S. pneumoniae infection. Furthermore, we demonstrate that such a therapeutic intervention reverses infection-associated gliosis and demyelination in the absence of changes in CNS bacterial burden. Together, these results suggest that targeting SP/NK-1R interactions is a strategy worthy of further study for the treatment of microbially induced neuroinflammation.

Recombinant expression of homodimeric 660 kDa human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin.

Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex proteins that are currently difficult or impossible to express using traditional plant and non-plant-based host systems. To test this hypothesis, we transformed soybeans with a synthetic gene encoding human thyroglobulin (hTG)-a 660 kDa homodimeric protein that is widely used in the diagnostic industry for screening and detection of thyroid disease. In the absence of a recombinant system that can produce recombinant hTG, research and diagnostic grade hTG continues to be purified from cadaver and surgically removed thyroid tissue. These less-than-ideal tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified hTG is used in sensitive ELISA screens. In this study, we report the successful expression of recombinant hTG in soybean seeds. Authenticity of the soy-derived protein was demonstrated using commercial ELISA kits developed specifically for the detection of hTG in patient sera. Western analyses and gel filtration chromatography demonstrated that recombinant hTG and thyroid-purified hTG are biologically similar with respect to size, mass, charge and subunit interaction. The recombinant protein was stable over three generations and accumulated to ~1.5% of total soluble seed protein. These results support our hypothesis that soybeans represent a practical alternative to traditional host systems for the expression of large and complex proteins.

3,4-Methylenedioxymethamphetamine (MDMA) alters acute gammaherpesvirus burden and limits interleukin 27 responses in a mouse model of viral infection.

AIMS: To test whether 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") abuse might increase the susceptibility, or alter the immune response, to murine gammaherpesvirus 68 (HV-68) and/or bacterial lipopolysaccharide. METHODS: Groups of experimental and control mice were subjected to three day binges of MDMA, and the effect of this drug abuse on acute and latent HV-68 viral burden were assessed. In vitro and in vivo studies were also performed to assess the MDMA effect on IL-27 expression in virally infected or LPS-exposed macrophages and dendritic cells, and latently infected animals, exposed to this drug of abuse. RESULTS: Acute viral burden was significantly increased in MDMA-treated mice when compared to controls. However the latent viral burden, and physiological and behavioral responses were not altered in infected mice despite repeated bingeing with MDMA. MDMA could limit the IL-27 response of HV-68 infected or LPS-exposed macrophages and dendritic cells in vitro and in vivo, demonstrating the ability of this drug to alter normal cytokine responses in the context of a viral infection and/or a TLR4 agonist. CONCLUSION: MDMA bingeing could alter the host's immune response resulting in greater acute viral replication and reductions in the production of the cytokine, IL-27 during immune responses.

Oral vaccine formulations stimulate mucosal and systemic antibody responses against staphylococcal enterotoxin B in a piglet model.

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.

Interleukin-27 expression following infection with the murine gammaherpesvirus 68.

IL-27 is a heterodimeric cytokine composed of p28 and Epstein Barr virus induced gene 3 (Ebi3) protein subunits. In the present study, we questioned whether murine gammaherpesvirus 68 (HV-68) could induce expression of Ebi3, p28, and IL-27 in this mouse model of an EBV-like infection. Cultured macrophages and dendritic cells exposed to HV-68 upregulated p28 mRNA expression and increased secretion of the p28 and IL-27 (p28+Ebi3) proteins. B220(+) and CD11b(+) cells also upregulated p28 mRNA expression following in vivo infection with this virus. Surprisingly, no significant increases in p28 or IL-27 protein production were observed in vivo during the acute or mononucleosis phases of the disease. The possibility that HV-68-induced upregulation of p28 mRNA expression primed cells for IL-27 secretion was suggested by the ability of a TLR4 agonist to augment cytokine production. When cultured macrophages and dendritic cells were exposed to virus plus a suboptimal dose of LPS, increased levels of p28 protein expression were observed. More importantly, when latently infected mice were challenged with a sublethal dose of LPS, augmented p28 and IL-27 protein production occurred. Using a model of sepsis, mice latently infected with HV-68 had exaggerated p28 protein production when compared to mice that were singularly infected or subjected to cecal ligation and puncture. Taken together, these studies define expression of HV-68 induced IL-27, and suggest that mice latently infected with this gammaherpesvirus will have exaggerated responses when confronted with other stimuli capable of inducing this member of the IL-12 family of cytokines.

Infection with murine gammaherpesvirus 68 exacerbates inflammatory bowel disease in IL-10-deficient mice.

OBJECTIVE: We questioned whether infection with murine gammaherpesvirus 68 (HV-68) might exacerbate inflammatory bowel disease using mice deficient in IL-10 (IL-10-/-) as a model of developing colitis. METHODS: Groups of C57BL/6 mice and IL-10-/- mice were mock-treated or infected with HV-68. Two months following infection, mice were euthanized and a variety of parameters were measured to quantify the extent of inflammation and the presence of virus. Measurements included survival, body weight, splenomegaly, colonic disease scores, liver histopathology, viable bacteria in the liver, and splenic viral burden. RESULTS: IL-10-/- mice infected with HV-68 displayed reduced survival, lower body weights, increased splenomegaly, exacerbated colonic disease scores, increased numbers of viable bacteria in the liver, and increased leukocyte liver infiltration when compared to mock-treated IL-10-/- mice or HV-68 infected C57BL/6 mice. Surprisingly, levels of infectious or latent virus were not significantly different between the groups of mice exposed to HV-68. CONCLUSIONS: The presence of HV-68 in IL-10-/- mice exacerbates the developing clinical disease in this animal model of colitis.

Neurogenic exacerbation of microglial and astrocyte responses to Neisseria meningitidis and Borrelia burgdorferi.

Although glial cells are recognized for their roles in maintaining neuronal function, there is growing appreciation of the ability of resident CNS cells to initiate and/or augment inflammation following trauma or infection. The tachykinin, substance P (SP), is well known to augment inflammatory responses at peripheral sites and its presence throughout the CNS raises the possibility that this neuropeptide might serve a similar function within the brain. In support of this hypothesis, we have recently demonstrated the expression of high affinity receptors for SP (Neurokinin-1 (NK-1) receptors) on microglia and shown that this tachykinin can significantly elevate bacterially induced inflammatory prostanoid production by isolated cultures of these cells. In the present study, we demonstrate that endogenous SP/NK-1R interactions are an essential component in the initiation and/or progression of CNS inflammation in vivo following exposure to two clinically relevant bacterial CNS pathogens, Neisseria meningitidis and Borrelia burgdorferi. We show that in vivo elevations in inflammatory cytokine production and decreases in the production of an immunosuppressive cytokine are markedly attenuated in mice genetically deficient in the expression of the NK-1R or in mice treated with a specific NK-1R antagonist. Furthermore, we have used isolated cultures of microglia and astrocytes to demonstrate that SP can augment inflammatory cytokine production by these resident CNS cell types following exposure to either of these bacterial pathogens. Taken together, these studies indicate a potentially important role for neurogenic exacerbation of resident glial immune responses in CNS inflammatory diseases, such as bacterial meningitis.

Ecstasy (3,4-methylenedioxymethamphetamine) limits murine gammaherpesvirus-68 induced monokine expression.

While Ecstasy (3,4-methylenedioxymethamphetamine, MDMA) has been shown to modulate immune responses, no studies have addressed drug-induced alterations to viral infection. In this study, bone marrow-derived macrophages were exposed to MDMA, then infected with murine gammaherpesvirus-68, and the expression of monokines assessed. MDMA-induced reductions in virus-stimulated monokine mRNA expression were observed in a dose-dependent manner. In particular, IL-6 mRNA expression and secretion was significantly decreased in gammaherpesvirus-infected macrophages exposed to MDMA. Concentrations of MDMA capable of reducing monokine production did not induce significant cell death and allowed normal viral gene expression. These studies represent the first to demonstrate the ability of this drug of abuse to alter a viral-induced macrophage response.

Expression of neuronal trace amine-associated receptor (Taar) mRNAs in leukocytes.

Trace amines such as tyramine, octopamine and beta-phenylethylamine bind with high affinity to the mammalian trace amine-associated receptor 1 (Taar1), potentially activating G-proteins in the synaptic membranes of target neurons. Recently there has been significant interest in Taar1, since this receptor can bind certain psychoactive drugs of abuse such as Ecstasy (3,4-methylenedioxymethamphetamine). Surprisingly, Ecstasy has been shown to alter responses of immune cells, and we questioned whether Taar receptors might be responsible for this effect. Using sensitive and quantitative RT-PCR assays, we found no detectable expression of Taar mRNA in bone marrow, or in primary cultures of mouse macrophages and dendritic cells whether quiescent or activated by exposure to lipopolysaccharide or the mouse gamma herpesvirus-68 (gammaHV-68). Mouse B cells and NK cells isolated from spleen, however, showed expression of several Taar mRNA species. Taar mRNA expression was also upregulated in human peripheral blood lymphocytes following in vitro stimulation with PHA. These studies represent the first to define expression of the mRNAs encoding these trace amine receptors in leukocytes.