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Type 1 diabetes (T1D) is an autoimmune disease characterized by T and B cell responses to proteins expressed by insulin-producing pancreatic β cells, inflammatory lesions within islets (insulitis), and β cell loss. We previously showed that Ag-specific tolerance targeting single β cell protein epitopes is effective in preventing T1D induced by transfer of monospecific diabetogenic CD4 and CD8 transgenic T cells to NOD.scid mice. However, tolerance induction to individual diabetogenic proteins, for example, GAD65 (glutamic acid decarboxylase 65) or insulin, has failed to ameliorate T1D both in wild-type NOD mice and in the clinic. Initiation and progression of T1D is likely due to activation of T cells specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant insulin, GAD65, and chromogranin A proteins were encapsulated within poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (COUR CNPs) to assess regulatory T cell induction, inhibition of Ag-specific T cell responses, and blockade of T1D induction/progression in NOD mice. Whereas treatment of NOD mice with CNPs containing a single protein inhibited the corresponding Ag-specific T cell response, inhibition of overt T1D development only occurred when all three diabetogenic proteins were included within the CNPs (CNP-T1D). Blockade of T1D following CNP-T1D tolerization was characterized by regulatory T cell induction and a significant decrease in both peri-insulitis and immune cell infiltration into pancreatic islets. As we have recently published that CNP treatment is both safe and induced Ag-specific tolerance in a phase 1/2a celiac disease clinical trial, Ag-specific tolerance induced by nanoparticles encapsulating multiple diabetogenic proteins is a promising approach to T1D treatment.

In this study, we evaluated the ability of negatively charged bio-degradable nanoparticles, ONP- 302, to inhibit tumor growth. Therapeutic treatment with ONP-302 in vivo resulted in a marked delay in tumor growth in three different syngeneic tumor models in immunocompetent mice. ONP- 302 efficacy persisted with depletion of CD8+ T cells in immunocompetent mice and also was effective in immune deficient mice. Examination of ONP-302 effects on components of the tumor microenvironment (TME) were explored. ONP-302 treatment caused a gene expression shift in TAMs toward the pro-inflammatory M1 type and substantially inhibited the expression of genes associated with the pro-tumorigenic function of CAFs. ONP-302 also induced apoptosis in CAFs in the TME. Together, these data support further development of ONP-302 as a novel first-in- class anti-cancer therapeutic that can be used as a single-agent as well as in combination therapies for the treatment of solid tumors due to its ability to modulate the TME.

Older people exhibit dysregulated innate immunity to respiratory viral infections, including influenza and SARS-CoV-2, and show an increase in morbidity and mortality. Nanoparticles are a potential practical therapeutic that could reduce exaggerated innate immune responses within the lungs during viral infection. However, such therapeutics have not been examined for effectiveness during respiratory viral infection, particular in aged hosts. Here, we employed a lethal model of influenza viral infection in vulnerable aged mice to examine the ability of biodegradable, cargo-free nanoparticles, designated ONP-302, to resolve innate immune dysfunction and improve outcomes during infection. We administered ONP-302 via i.v. injection to aged mice at day 3 after infection, when the hyperinflammatory innate immune response was already established. During infection, we found that ONP-302 treatment reduced the numbers of inflammatory monocytes within the lungs and increased their number in both the liver and spleen, without impacting viral clearance. Importantly, cargo-free nanoparticles reduced lung damage, reduced histological lung inflammation, and improved gas exchange and, ultimately, the clinical outcomes in influenza-infected aged mice. In conclusion, ONP-302 improves outcomes in influenza-infected aged mice. Thus, our study provides information concerning a practical therapeutic, which, if translated clinically, could improve disease outcomes for vulnerable older patients suffering from respiratory viral infections.

e14552 Background: Recent advances in the field of cancer immunology have highlighted the importance of the immune system for eliminating tumors. Numerous studies have shown that tumor-infiltrating immune cells such as antigen-presenting cells (APCs), T cells, and natural killer (NK) cells play critical roles in tumor control. However, the inflammatory anti-tumor immune response is counteracted by the induction of immune regulatory mechanisms within the tumor microenvironment (TME). These findings have led to the development of immune-targeted therapies, which are aimed at activating anti-tumor immune signaling pathways and enhancing anti-tumor immune function. While immunotherapies, have revolutionized the treatment of several solid tumors and leukemias, at best response rates remain low at 25%-30%, and a portion of patients eventually develop resistance to therapy leading to disease progression and mortality. Methods: We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic- co-glycolic acid) (PLGA) nanoparticles, ONP-302, free from drugs or other bioactive agents, for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302 nanoparticles, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. The efficacy of ONP-302 nanoparticles at inducing an anti-tumor immune response was evaluated using syngeneic mouse tumor models. Results: ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. Conclusions: These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

e14553 Background: The tumor microenvironment (TME) plays a crucial role in tumor growth and progression and has a significant influence on response to therapy. The TME consists of myeloid-derived cells, stroma (e.g. fibroblasts and extracellular matrix (ECM)), and the vasculature that together support tumor growth and progression. Studies in animal models and in humans show that myeloid- derived cells such as myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) engage in activities that support tumor growth and progression. These cells also promote immune suppression in the TME that blunts the efficacy of the anti-cancer drugs and immune-targeted therapies such as immune checkpoint inhibitors. In addition to MDSCs, cancer-associated fibroblasts (CAFs) in the TME support tumor progression via production of pro-tumor and pro-angiogenic growth-factors, remodeling of the ECM via production of proteases, and suppression of anti-tumor immune function. CAF abundance in the TME is a negative prognostic factor for several solid tumors and is associated with negative outcomes and poor response to immune-targeted therapies like immune checkpoint inhibitors. ONP-302 nanoparticles fabricated from biodegradable poly (lactic-co-glycolic acid)(PLGA) polymer have been previously described in the literature for the treatment of acute inflammatory conditions via immuno-modulatory effects on myeloid derived cells. Here, we evaluated the efficacy of ONP-302 nanoparticles at inhibiting tumor growth via targeted inhibition of myeloid-derived cells and reshaping of the TME. Methods: ONP-302 anti-tumor efficacy was evaluated in syngeneic mouse tumor models using both immunocompetent and immunodeficient mice. We examined the effect of ONP-302 treatment tumor growth kinetics and effects on the major cellular constituents of the TME such as myeloid-derived cells and CAFs. Results: Therapeutic treatment with ONP-302 in vivo resulted in a marked delay in tumor growth in three different syngeneic tumor models in immunocompetent mice. ONP- 302 efficacy persisted with depletion of CD8+ T cells in immunocompetent mice and also was effective in immune deficient mice. We found ONP-302 treatment caused a gene expression shift in TAMs toward the pro-inflammatory M1 type and substantially inhibited the expression of genes associated with the pro-tumorigenic function of CAFs. ONP-302 also induced apoptosis in CAFs in the TME. Conclusions: Our data indicate that the slowing of tumor growth after ONP-302 treatment is due to disruptions in known signaling pathways involving TAMs and CAFs, pathways typically supporting tumor growth. These data taken in concert indicate the activity of ONP-302 is pleotropic and affects multiple pathways.

e14555 Background: Metastasis is responsible for most cancer-related deaths and depends on the formation of a metastatic niche at distal sites. The metastatic niche consists primarily of immunosuppressive myeloid cells, such as neutrophils and monocytes. These cells are recruited to distal sites in response to cancer-induced immune dysregulation and promote the recruitment and survival of disseminated tumor cells. We have employed drug-free poly(lactide-co-glycolide) (PLGA) nanoparticles, ONP-302, to modulate myeloid cell phenotypes and trafficking to disrupt the formation of the metastatic niche. The anti-metastatic efficacy of ONP-302 nanoparticles was evaluated in a translationally relevant mouse model of orthotopic triple-negative breast cancer. Methods: ONP-302 nanoparticles were evaluated in the syngeneic 4T1 model of triple-negative breast cancer in female BALB/c or RAG1KO BALB/c mice. ONP-302 nanoparticles intravenously injected into tumor-bearing mice every 3 days starting 1 day after tumor inoculation. Lungs were extracted 14 and 21 days after inoculation for analysis by single cell RNA sequencing and quantification of metastases by luminescent imaging, respectively. In resection studies, primary tumors were resected 11 days after inoculation and tissues were extracted 42 days after resection for quantification of metastases. Results: ONP-302 nanoparticles significantly reduced primary tumor growth and completely inhibited the formation of lung metastases. ONP-302 was also effective at suppressing metastasis when applied following surgical resection. In mice which received ONP-302 after surgical resection, lung metastases were undetectable up to 42 days after resection, while 44% of control mice developed metastases. These findings suggest that ONP-302 targets both primary tumor growth and metastatic colonization. Single cell RNA sequencing was employed to analyze ONP-302 modulation of the lung metastatic niche. The lungs of ONP-302 treated mice had lower neutrophil infiltration and a higher proportion of monocytes and dendritic cells compared to control mice. Gene expression pathways associated with inflammatory interferon and tumor necrosis factor responses were upregulated in neutrophils, monocytes, and dendritic cells from ONP-302 treated mice. The upregulation of these pathways suggests that ONP-302 treatment prevents the formation of an immunosuppressive niche and promotes anti-tumor cytotoxicity. Adaptive immunity was essential for ONP-302 efficacy, as T and B cell deficient RAG1KO mice demonstrated complete loss of ONP-302 efficacy both in slowing primary tumor growth and preventing metastasis. Conclusions: Our findings demonstrate that our drug-free PLG nanoparticles, ONP-302, impede cancer metastasis as a monotherapy by inhibiting the accumulation of immunosuppressive myeloid cells.

Type 1 diabetes (T1D) is an autoimmune disease characterized by inflammatory lesions within islets (insulitis) and β cell loss which is associated T cell and B cell responses specific for pancreatic islet β cell proteins. Data show that treatment with poly(lactide-co-glycolide) (PLGA) nanoparticles (i.e. TIMP/COUR’s CNP) containing a single diabetogenic peptide blocks T1D induced by transfer of CD4+ BDC2.5 or CD8+ NY8.3 TCR transgenic T cells to NOD-scid mice. In contrast, Ag-specific tolerance approaches targeting single β-islet cell protein epitopes, e.g. GAD65 or Insulin, have failed to achieve efficacy in both wildtype NOD mice and clinical trials. The possibility exists that T1D initiation and progression is due to activation of T cell populations specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant Chormogranin A, GAD65, and Insulin proteins were encapsulated within CNPs to assess Treg/Tr1 cell induction, inhibition of Ag-specific T cell responses, and blockade of T1D in NOD mice. While treatment of NOD mice with CNPs containing a single protein inhibited the corresponding Ag-specific T cell response, the inhibition of T1D development only occurred if all three diabetogenic proteins were included within the CNPs (CNP-T1D). CNP-T1D-induced blockade of T1D was characterized by Treg/Tr1 cell induction and a significant decrease in both peri-insulitis and immune cell infiltration into pancreatic islets. We have recently published that CNP treatment is both safe and induces Ag-specific tolerance in Phase I/IIa celiac disease clinical trials. Therefore, utilization of this technology including multiple diabetogenic proteins is a promising Ag-specific tolerance approach to T1D treatment.

Older people exhibit dysregulated innate immunity to respiratory viral infections, including influenza and SARS-CoV-2, to increase morbidity and mortality. Nanoparticles are a potential practical therapeutic that could divert exaggerated innate immune responses away from the lungs during viral infection. However, such therapeutics have not been examined for effectiveness during respiratory viral infection, particular in aged hosts. Here, we employed a highly lethal model of influenza viral infection in vulnerable aged mice to examine the ability of biodegradable, cargo-free nanoparticles, designated ONP-302, to resolve innate immune dysfunction and improve outcomes during infection. We administered ONP-302 via intravenous injection to aged mice at day 3 post-infection when the hyperinflammatory innate immune response is already established. We found that ONP-302 treatment diverted tissue-damaging inflammatory monocytes from the lungs to the spleen without impacting viral clearance or reducing chemo-attractive signals in the infected lung. Importantly, cargo-free nanoparticles reduced lung damage and histological lung inflammation and improved gas exchange and, ultimately, the clinical outcomes in influenza-infected aged mice. ONP-302 improves outcomes in influenza-infected, vulnerable, aged mice. Thus, our study provides vital fundamental information concerning a practical therapeutic which, if translated clinically, could improve disease outcomes for vulnerable older patients suffering from respiratory viral infections. Supported by COUR Pharmaceuticals

There are many autoimmune diseases where pathologic self T cells are the underlying cause of disease symptoms and progression. T cells are found at the site of tissue destruction, resulting not only in the exacerbation of disease, but also the release of self-epitopes in the context of inflammation resulting in the activation of additional T cell populations perpetuating disease. Published data have shown treatment with antigen-containing biodegradablepoly(lactide-co-glycolide) (PLGA) nanoparticles, i.e. tolerogenic immune-modifying particles (TIMP), is both safe and induces antigen-specific tolerance in mouse models of autoimmunity and allergy, as well as in a celiac disease Phase I/IIa clinical trial. This study further investigated the role and mechanism of TIMP-induced tolerance. The present data show that TIMP treatment modulated T cells specific for spread epitopes associated with disease progression, but not encapsulated within the TIMP, i.e. tissue-specific bystander suppression. The PLP139–151 TCR transgenic model systems was utilized to determine the cellular and molecular mechanisms driving antigen specific tolerance mediated by tolerogenic nanoparticle treatment. These data show antigen-specific TIMP treatment induced both FoxP3+ iTregs and IL-10+ Tr1 regulatory phenotypes within the antigen-specific T cell populations in both naïve mice and mice pre-primed with antigen/CFA. Additionally, both functional Tregs and IL-10 are required for TIMP-induced tolerance. Treatment thus activates various antigen-specific Treg subsets capable of regulating responses to disease-relevant autoepitopes not encapsulated within the TIMP via release of immunoregulatory cytokines within the inflammatory site. Supported by funding provided by Cour Pharmaceuticals Development, Inc.

BACKGROUND & AIMS: In celiac disease (CeD), gluten induces immune activation, leading to enteropathy. TAK-101, gluten protein (gliadin) encapsulated in negatively charged poly(dl-lactide-co-glycolic acid) nanoparticles, is designed to induce gluten-specific tolerance. METHODS: TAK-101 was evaluated in phase 1 dose escalation safety and phase 2a double-blind, randomized, placebo-controlled studies. Primary endpoints included pharmacokinetics, safety, and tolerability of TAK-101 (phase 1) and change from baseline in circulating gliadinspecific interferon-γ–producing cells at day 6 of gluten challenge, in patients with CeD (phase 2a). Secondary endpoints in the phase 2a study included changes from baseline in enteropathy (villus height to crypt depth ratio [Vh:Cd]), and frequency of intestinal intraepithelial lymphocytes and peripheral gut-homing T cells. RESULTS: In phase 2a, 33 randomized patients completed the 14-day gluten challenge. TAK-101 induced an 88% reduction in change from baseline in interferon-γ spot-forming units vs placebo (2.01 vs 17.58, P = .006). Vh:Cd deteriorated in the placebo group (−0.63, P = .002), but not in the TAK-101 group (−0.18, P = .110), although the intergroup change from baseline was not significant (P = .08). Intraepithelial lymphocyte numbers remained equal. TAK-101 reduced changes in circulating α4β7(+)CD4(+) (0.26 vs 1.05, P = .032), αEβ7(+)CD8(+) (0.69 vs 3.64, P = .003), and γδ (0.15 vs 1.59, P = .010) effector memory T cells. TAK-101 (up to 8 mg/kg) induced no clinically meaningful changes in vital signs or routine clinical laboratory evaluations. No serious adverse events occurred. CONCLUSIONS: TAK-101 was well tolerated and prevented gluten-induced immune activation in CeD. The findings from the present clinical trial suggest that antigen-specific tolerance was induced and represent a novel approach translatable to other immune-mediated diseases. ClinicalTrials.gov identifiers: NCT03486990 and NCT03738475.

In this study, we show that a negatively charged biodegradable nanoparticles, CNP-301, have strong anti-tumor activity in several tumor models. This effect appears to be driven by shifting polarization of tumor associated macrophages (TAMs) to an anti-tumor M1 type and by downregulation of pro-tumorigenic cancer associated fibroblasts. Moreover, we show that CNP-301 treatment induces apoptosis of cancer associated fibroblasts (CAFs) in the tumor supporting a mechanism where perturbations of the TME and the associated stroma results in anti-tumor effects.

Chemokines are a superfamily of chemotactic cytokines that play an important role in leukocyte trafficking and have been implicated as functional mediators of immunopathology in experimental autoimmune encephalomyelitis (EAE). In the present study, we investigated the role of the CCL20 receptor, CCR6, in chronic EAE. After immunization with myelin oligodendrocyte glycoprotein 35-55 in CFA, CCR6(-/-) mice developed a significantly more severe chronic EAE as compared to wild type immunized animals. CCR6 expression was not required by T cells to induce EAE. Measurement of peripheral T cell responses showed differences in IFN-gamma and IL-17 responses between CCR6(-/-) and wild type mice. At the time when CCR6(-/-) mice showed significantly more severe chronic EAE there was a significant decrease in PD-L1-expressing mDC in the spleens and no differences in Foxp3 Treg. Furthermore, add back of mDC with increased PD-L1 expression to CCR6(-/-) mice reduced the severe chronic EAE disease phase to that of wild type controls. The results suggest a role for CCR6-expressing PDL1(+) mDC in regulating EAE progression.

Experimental autoimmune encephalomyelitis is a T cell-mediated demyelinating disease of the CNS that serves as a model for the human disease multiple sclerosis. Increased expression of the chemokine CCL2 in the CNS has been demonstrated to be important in the development of demyelinating disease presumably by attracting inflammatory cells. However, the mechanism of how CCL2 regulates disease pathogenesis has not been fully elucidated. Using radiation bone marrow chimeric mice we demonstrated that optimum disease was achieved when CCL2 was glia derived. Furthermore, CNS production of CCL2 resulted in the accumulation of iNOS-producing CD11b+CD11c+ dendritic cells and TNF-producing macrophages important for demyelination. Lack of glial-derived CCL2 production did not influence experimental autoimmune encephalomyelitis by altering either Th1 or Th17 cells, as there were no differences in these populations in the CNS or periphery between groups. These results demonstrate that the glial-derived CCL2 is important for the attraction of TNF- and iNOS-producing dendritic cells and effector macrophages to the CNS for development of subsequent autoimmune disease.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a murine model for multiple sclerosis, involves recruitment of T cells and macrophages to the CNS after infection. We hypothesized that CCR2, the only known receptor for CCL2, would be required for TMEV-induced demyelinating disease development because of its role in macrophage recruitment. TMEV-infected SJL CCR2 knockout (KO) mice showed decreased long-term clinical disease severity and less demyelination compared with controls. Flow cytometric data indicated that macrophages (CD45(high) CD11b(+) ) in the CNS of TMEV-infected CCR2 KO mice were decreased compared with control mice throughout disease. CD4(+) and CD8(+) T cell percentages in the CNS of TMEV-infected control and CCR2 KO mice were similar over the course of disease. There were no apparent differences between CCR2 KO and control peripheral immune responses. The frequency of interferon-gamma-producing T cells in response to proteolipid protein 139-151 in the CNS was also similar during the autoimmunity stage of TMEV-induced demyelinating disease. These data suggest that CCR2 is important for development of clinical disease by regulating macrophage accumulation after TMEV infection.

CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, a member of the CC chemokine family, is a chemoattractant for monocytes and T cells through interaction with its receptor CCR2. In the present study, we examined a T helper cell type 1 (Th1)-dependent disease, proteolipid protein-induced experimental autoimmune encephalomyelitis, in a transgenic mouse line that constitutively expressed low levels of CCL2 in the central nervous system (CNS) under control of the astrocyte-specific glial fibrillary acidic protein promoter. CCL2 transgenic mice developed significantly milder clinical disease than littermate controls. As determined by flow cytometry, mononuclear cell infiltrates in the CNS tissues of CCL2 transgenic and littermate-control mice contained equal numbers of CD4+ and CD8+ T cells, and the CCL2 transgenic mice showed an enhanced number of CNS-infiltrating monocytes. CNS antigen-specific T cells from CCL2 transgenic mice produced markedly less interferon-γ. Overexpression of CCL2 in the CNS resulted in decreased interleukin-12 receptor expression by antigen-specific T cells. Collectively, these results indicate that sustained, tissue-specific expression of CCL2 in vivo down-regulates the Th1 autoimmune response, culminating in milder clinical disease.

Expression of high affinity IL-12 receptors is required for IL-12-mediated IFN-γ production. Activation of this pathway has been shown to be critical in generating optimal cell-mediated immunity. Therefore, increased IL-12 receptor expression might be expected in the host response after infection by an intracellular bacterial pathogen. In the present study, we have made the surprising discovery that infection with Salmonella results in an early reduction of high affinity IL-12 receptor expression and activation. After oral inoculation with Salmonella, the level of mRNA expression encoding IL-12 receptor β2 (IL-12Rβ2) subunit was diminished 12 h postinfection in the mesenteric lymph nodes and subsequently in the spleen. Furthermore, decreased IL-12Rβ2 mRNA expression was observed in CD4+ T lymphocytes isolated from the mesenteric lymph nodes and spleens of infected mice. Attenuated IL-12Rβ2 mRNA expression correlated with reduced receptor signaling, as demonstrated by reduced IL-12-induced STAT4 phosphorylation in enriched T lymphocytes isolated from the mesenteric lymph nodes and spleens of Salmonella-infected mice. These in vivo results were substantiated with an in vitro model system. In this model system, T lymphocytes cocultured with Salmonella-infected macrophages expressed less IL-12Rβ2 mRNA. The cocultured T cells were also less responsive to IL-12 as assessed by reduced phosphorylation of STAT4 and limited IFN-γ secretion. Together, these studies suggest that Salmonella can limit an optimal host immune response by reducing the expression and activity of high affinity IL-12 receptors.

Professional antigen presenting cells, such as macrophages, can be activated by intracellular calcium-dependent as well as calcium-independent mechanisms, depending upon the stimulus used. In this report, we addressed the mechanism of substance P-induced intracellular signalling in murine macrophages and dendritic cells. While no increases in intracellular calcium concentration were detected in macrophages or dendritic cells using sensitive fluorimetric techniques, substance P did induce rapid enhanced activation of NF-κB, a transcriptional activator known to regulate pro-inflammatory cytokines. These data provide an important mechanism by which substance P may augment the production of pro-inflammatory molecules.

Optimal immune responses against an intracellular bacterial pathogen, such as Salmonella , involve the production of gamma interferon (IFN-γ), which activates macrophages. It has recently been suggested that, interleukin-18 (IL-18), in addition to IL-12, contributes to the induction of IFN-γ following infection. Given this hypothesis, an optimal host immune response against intracellular bacterial pathogens would include the induction of IL-18 secretion by macrophages due to Salmonella infection. We questioned whether Salmonella could induce macrophages to upregulate their expression of IL-18 mRNA and secretion of IL-18. With cultures of murine macrophages, we were surprised to find that infection by wild-type Salmonella dublin resulted in decreased expression of IL-18 mRNA and IL-18 secretion rather than an increase. Reduction of macrophage-derived IL-18 expression by wild-type Salmonella occurred early in the response, suggesting a direct effect. Furthermore, mice orally inoculated with wild-type Salmonella were shown to have reduced IL-18 mRNA expression at mucosal sites within hours postinoculation. Together these studies demonstrate Salmonella -induced reductions in IL-18 expression, suggesting that this intracellular pathogen may be capable of limiting a potentially protective immune response.

Transposition of the maize Suppressor–mutator ( Spm ) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range.

The immunologic privilege of the central nervous system (CNS) makes it crucial that CNS resident cells be capable of responding rapidly to infection. Astrocytes have been reported to express Toll-like receptors (TLRs), hallmark pattern recognition receptors of the innate immune system, and respond to their ligation with cytokine production. Astrocytes have also been reported to respond to cytokines of the adaptive immune system with the induction of antigen presentation functions. Here we have compared the ability of TLR stimuli and the adaptive immune cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to induce a variety of immunologic functions of astrocytes. We show that innate signals LPS- and poly I:C lead to stronger upregulation of TLRs and production of the cytokines IL-6 and TNF-alpha as well as innate immune effector molecules IFN-alpha4, IFN-beta, and iNOS compared with cytokine-stimulated astrocytes. Both innate stimulation and adaptive stimulation induce similar expression of the chemokines CCL2, CCL3, and CCL5, as well as similar enhancement of adhesion molecule ICAM-1 and VCAM-1 expression by astrocytes. Stimulation with adaptive immune cytokines, however, was unique in its ability to induce upregulation of MHC II and the functional ability of astrocytes to activate CD4(+) T cells. These results indicate potentially important and changing roles for astrocytes during the progression of CNS infection.

Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated demyelinating disease of the central nervous system (CNS) that serves as a model for multiple sclerosis (MS). Insights into the pathogenesis of this model may help scientists understand the human disease and aid in rational drug discovery. In this review we summarize the role of chemokines and chemokine receptors in disease pathogenesis and suggest a pathway of events that leads to demyelination and subsequent clinical disease manifestation.

Staphylococcus aureusinvades osteoblasts and can persist in the intracellular environment. The present study examined the role of osteoblast mitogen-activated protein kinase (MAPK) pathways in bacterial invasion.S. aureusinfection of normal human and mouse osteoblasts resulted in an increase in the phosphorylation of the extracellular signal-regulated protein kinases (ERK 1 and 2). This stimulation of ERK 1 and 2 correlated with the time course ofS. aureusinvasion, and bacterial adherence induced the MAPK pathway. ERK 1 and 2 phosphorylation was time and dose dependent and required activeS. aureusgene expression for maximal induction. The nonpathogenicStaphylococcus carnosuswas also able to induce ERK 1 and 2 phosphorylation, albeit at lower levels thanS. aureus. Phosphorylation of the stress-activated protein kinases was increased in both infected human and mouse osteoblasts; however, the p38 MAPK pathway was not activated in response toS. aureus. Finally, the transcription factor c-Jun, but not Elk-1 or ATF-2, was phosphorylated in response toS. aureusinfection.

Activation of microglia and astrocytes in experimental autoimmune encephalitis (EAE) has been associated with chemokine induction in the CNS resulting in attraction of inflammatory infiltrate. The role of IL-17 in autoimmune disease has called into question the role of IFN-γ in EAE. In this report we investigate the necessity of IFN-γ receptor signaling in disease induction. To test this we made IFN-γR−/− radiation bone chimeras with animals expressing IFN-γR in the CNS, animals deficient for IFN-γR in the CNS but able to signal in the periphery and a wild-type control (WT). We found IFN-γR expression on astrocytes and microglia was critical for clinical disease symptoms in the adoptive transfer model of EAE. Without IFN-γ activation in the CNS there were no infiltrating leukocytes while in WT and in animals capable of signaling in the CNS there were CD4, CD8, and CD11b infiltrating cells. CCL2, CCL5 and CXCL10 were significantly decreased in CNS homogenates of animals devoid of IFN-γR on astrocytes and microglia compared to WT and mice expressing IFN-γR on peripheral leukocytes. These results argue that IFN-γ activation in the CNS is essential for clinical EAE disease induction. Supported by NIH R01 NS034510.