Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Cathepsin W, T-cell receptor-associated transmembrane adapter 1, lymphotactin and killer cell lectin like receptor K1 are sensitive and specific RNA biomarkers of canine epitheliotropic lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an uncommon type of lymphoma involving malignant skin-resident or skin-homing T cells. Canine epitheliotropic lymphoma (EL) is the most common form of CTCL in dogs, and it also spontaneously arises from T lymphocytes in the mucosa and skin. Clinically, it can be difficult to distinguish early-stage CTCLs apart from other forms of benign interface dermatitis (ID) in both dogs and people. Our objective was to identify novel biomarkers that can distinguish EL from other forms of ID, and perform comparative transcriptomics of human CTCL and canine EL. Here, we present a retrospective gene expression study that employed archival tissue from biorepositories. We analyzed a discovery cohort of 6 canines and a validation cohort of 8 canines with EL which occurred spontaneously in client-owned companion dogs. We performed comparative targeted transcriptomics studies using NanoString to assess 160 genes from lesional skin biopsies from the discovery cohort and 800 genes from the validation cohort to identify any significant differences that may reflect oncogenesis and immunopathogenesis. We further sought to determine if gene expression in EL and CTCL are conserved across humans and canines by comparing our data to previously published human datasets. Similar chemokine profiles were observed in dog EL and human CTCL, and analyses were performed to validate potential biomarkers and drivers of disease. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CD244, CXCL10, and CCL5 in EL in dogs compared to healthy controls. Importantly, CTSW, TRAT1 and KLRK1 distinguished EL from all other forms of interface dermatitis we studied, providing much-needed biomarkers for the veterinary field. XCL1/XCL2 were also highly specific of EL in our validation cohort. Future studies exploring the oncogenesis of spontaneous lymphomas in companion animals will expand our understanding of these disorders. Biomarkers may be useful for predicting disease prognosis and treatment responses. We plan to use our data to inform future development of targeted therapies, as well as for repurposing drugs for both veterinary and human medicine.

EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes.

NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT-regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma-xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth. SIGNIFICANCE: Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827.

EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes.

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUT's ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCE: Identification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

Using Gene Expression Analysis to Understand Complex Autoimmune Skin Disease Patients: A Series of Four Canine Cutaneous Lupus Erythematosus Cases.

Cutaneous Lupus Erythematosus (CLE) is an autoimmune skin disease that occurs in almost two-thirds of people with Systemic Lupus Erythematosus (SLE) and can exist as its own entity. Despite its negative impact on the quality of life of patients, lupus pathogenesis is not fully understood. In recent years, the role of gene expression analysis has become important in understanding cellular functions and disease causation within and across species. Interestingly, dogs also develop CLE, providing a spontaneous animal model of disease. Here, we present a targeted transcriptomic analysis of skin biopsies from a case series of four dogs with complex autoimmunity with suspected CLE. We identified 92 differentially expressed genes (DEGs), including type 1 interferon, B cell, and T cell-related genes, in the four cases compared to healthy skin margin controls. Additionally, we compared our results with existing CLE datasets from humans and mice and found that humans and canines share 49 DEGs, whereas humans and mice shared only 25 DEGs in our gene set. Immunohistochemistry of IFNG and CXCL10, two of the most highly upregulated inflammatory mediators, confirmed protein-level expression and revealed immune cells as the primary source of CXCL10 in dogs with SLE, whereas keratinocytes stained strongly for CXCL10 in dogs without SLE. We propose that gene expression analysis may aid the diagnosis of complex autoimmune skin diseases and that dogs may provide important insights into CLE and SLE pathogeneses, or more broadly, skin manifestations during systemic autoimmunity.

Gene Expression Analysis in Four Dogs With Canine Pemphigus Clinical Subtypes Reveals B Cell Signatures and Immune Activation Pathways Similar to Human Disease.

Pemphigus is a group of autoimmune-mediated mucocutaneous blistering diseases characterized by acantholysis. Pemphigus has also been recognized in dogs and shares similar clinical characteristics and variants with human pemphigus. While relationships between human and canine pemphigus have been reported, gene expression patterns across species have not been described in the literature. We sought to perform gene expression analysis of lesional skin tissue from four dogs with various forms of pemphigus to examine gene expression during spontaneous disease in dogs. We found increased T and B cell signatures in canine pemphigus lesions compared to controls, as well as significant upregulation of CCL3, CCL4, CXCL10, and CXCL8 (IL8), among other genes. Similar chemokine/cytokine expression patterns and immune infiltrates have been reported in humans, suggesting that these genes play a role in spontaneous disease. Direct comparison of our dataset to previously published human pemphigus datasets revealed five conserved differentially expressed genes: CD19, WIF1, CXCL10, CD86, and S100A12. Our data expands our understanding of pemphigus and facilitates identification of biomarkers for prediction of disease prognosis and treatment response, which may be useful for future veterinary and human clinical trials.

Limited TCR repertoire and ENTPD1 dysregulation mark late-stage COVID-19.

T cell exhaustion and dysfunction are hallmarks of severe COVID-19. To gain insights into the pathways underlying these alterations, we performed a comprehensive transcriptome analysis of peripheral-blood-mononuclear-cells (PBMCs), spleen, lung, kidney, liver, and heart obtained at autopsy from COVID-19 patients and matched controls, using the nCounter CAR-T-Characterization panel. We found substantial gene alterations in COVID-19-impacted organs, especially the lung where altered TCR repertoires are noted. Reduced TCR repertoires are also observed in PBMCs of severe COVID-19 patients. ENTPD1/CD39, an ectoenzyme defining exhausted T-cells, is upregulated in the lung, liver, spleen, and PBMCs of severe COVID-19 patients where expression positively correlates with markers of vasculopathy. Heightened ENTPD1/CD39 is paralleled by elevations in STAT-3 and HIF-1α transcription factors; and by markedly reduced CD39-antisense-RNA, a long-noncoding-RNA negatively regulating ENTPD1/CD39 at the post-transcriptional level. Limited TCR repertoire and aberrant regulation of ENTPD1/CD39 could have permissive roles in COVID-19 progression and indicate potential therapeutic targets to reverse disease.

Targeted Transcriptomic Analysis of C57BL/6 and BALB/c Mice During Progressive Chronic Toxoplasma gondii Infection Reveals Changes in Host and Parasite Gene Expression Relating to Neuropathology and Resolution.

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection.

SPARC coordinates extracellular matrix remodeling and efficient recruitment to and migration of antigen-specific T cells in the brain following infection.

Central nervous system (CNS) injury and infection can result in profound tissue remodeling in the brain, the mechanism and purpose of which is poorly understood. Infection with the protozoan parasite Toxoplasma gondii causes chronic infection and inflammation in the brain parenchyma. Control of parasite replication requires the continuous presence of IFNγ-producing T cells to keep T. gondii in its slowly replicating cyst form. During infection, a network of extracellular matrix fibers, revealed using multiphoton microscopy, forms in the brain. The origin and composition of these structures are unknown but the fibers have been observed to act as a substrate for migrating T cells. In this study, we show a critical regulator of extracellular matrix (ECM) remodeling, Secreted Protein, Acidic, Rich in Cysteine (SPARC), is upregulated in the brain during the early phases of infection in the frontal cortex. In the absence of SPARC, a reduced and disordered fibrous network, increased parasite burden, and reduced antigen-specific T cell entry into the brain points to a role for SPARC in T cell recruitment to and migration within the brain. We also report SPARC can directly bind to CCR7 ligands CCL19 and CCL21 but not CXCL10, and enhance migration toward a chemokine gradient. Measurement of T cell behavior points to tissue remodeling being important for access of immune cells to the brain and facilitating cellular locomotion. Together, these data identify SPARC as an important regulatory component of immune cell trafficking and access to the inflamed CNS.

Shared inflammatory and skin-specific gene signatures reveal common drivers of discoid lupus erythematosus in canines, humans and mice.

Autoimmune skin diseases are complex and are thought to arise from a combination of genetics and environmental exposures, which trigger an ongoing immune response against self-antigens. Companion animals including cats and dogs are known to develop inflammatory skin conditions similar to humans and share the same environment, providing opportunities to study spontaneous disease that encompasses genetic and environmental factors with a One Health approach. A strength of comparative immunology approaches is that immune profiles may be assessed across different species to better identify shared or conserved pathways that might drive inflammation. Here, we performed a comparative study of skin from canine discoid lupus erythematosus (DLE) using NanoString nCounter technology. We compared these gene expression patterns to those of human DLE and a mouse model of cutaneous lupus. We found strong interferon signatures, with CXCL10, ISG15, and an S100 gene family member among the highest, most significant DEGs upregulated across species. Cell type analysis revealed marked T-cell and B-cell infiltration. Interestingly, canine DLE samples also recapitulated downregulated skin homeostatic genes observed in human DLE. We conclude that spontaneous DLE in dogs captures many features that are present in human disease and may serve as a more complete model for conducting further genomic and/or transcriptomic studies.

Case Series: Gene Expression Analysis in Canine Vogt-Koyanagi-Harada/Uveodermatologic Syndrome and Vitiligo Reveals Conserved Immunopathogenesis Pathways Between Dog and Human Autoimmune Pigmentary Disorders.

Vogt-Koyanagi-Harada syndrome (VKH) and vitiligo are autoimmune diseases that target melanocytes. VKH affects several organs such as the skin, hair follicle, eyes, ears, and meninges, whereas vitiligo is often limited to the skin and mucosa. Many studies have identified immune genes, pathways and cells that drive the pathogeneses of VKH and vitiligo, including interleukins, chemokines, cytotoxic T-cells, and other leukocytes. Here, we present case studies of 2 canines with VKH and 1 with vitiligo, which occurred spontaneously in client-owned companion dogs. We performed comparative transcriptomics and immunohistochemistry studies on lesional skin biopsies from these cases in order to determine if the immunopathogenesis of autoimmune responses against melanocytes are conserved. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CTSW, CXCL10, and CCL5 in both VKH and vitiligo in dogs compared to healthy controls. Similar findings were reported in humans, suggesting that these genes play a role in the pathogenesis of spontaneous VKH and vitiligo. T cell-associated genes, including FOXP3 and TBX21, were enriched, while IGFBP5, FOXO1, and PECAM1 were decreased compared to healthy controls. Further, we identified TGFB3, SFRP2, and CXCL7 as additional potential drivers of autoimmune pigmentary disorders. Future studies exploring the immunopathogenesis of spontaneous autoimmunity will expand our understanding of these disorders, and will be useful in developing targeted therapies, repurposing drugs for veterinary and human medicine, and predicting disease prognosis and treatment response.

A nanoparticle-incorporated STING activator enhances antitumor immunity in PD-L1-insensitive models of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has few therapeutic options, and alternative approaches are urgently needed. Stimulator of IFN genes (STING) is becoming an exciting target for therapeutic adjuvants. However, STING resides inside the cell, and the intracellular delivery of CDNs, such as cGAMP, is required for the optimal activation of STING. We show that liposomal nanoparticle-delivered cGAMP (cGAMP-NP) activates STING more effectively than soluble cGAMP. These particles induce innate and adaptive host immune responses to preexisting tumors in both orthotopic and genetically engineered models of basal-like TNBC. cGAMP-NPs also reduce melanoma tumor load, with limited responsivity to anti-PD-L1. Within the tumor microenvironment, cGAMP-NPs direct both mouse and human macrophages (M), reprograming from protumorigenic M2-like phenotype toward M1-like phenotype; enhance MHC and costimulatory molecule expression; reduce M2 biomarkers; increase IFN-γ-producing T cells; augment tumor apoptosis; and increase CD4+ and CD8+ T cell infiltration. Activated T cells are required for tumor suppression, as their depletion reduces antitumor activity. Importantly, cGAMP-NPs prevent the formation of secondary tumors, and a single dose is sufficient to inhibit TNBC. These data suggest that a minimal system comprised of cGAMP-NP alone is sufficient to modulate the tumor microenvironment to effectively control PD-L1-insensitive TNBC.

A robust microparticle platform for a STING-targeted adjuvant that enhances both humoral and cellular immunity during vaccination.

Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)-driven anti-viral immunity; however, a major hindrance is STING's cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3'3'-cGAMP (cGAMP), encapsulated in acid-sensitive acetalated dextran (Ace-DEX) polymeric microparticles (MPs) which passively target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace-DEX cGAMP MPs enhanced type-I interferon responses nearly 1000-fold in vitro and 50-fold in vivo, caused up to a 104-fold boost in antibody titers, increased Th1-associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post-immunization when using CDN adjuvant doses up to 100-fold lower than previous reports. For these reasons, Ace-DEX MP-encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity.

NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes.

Inflammation in the brain accompanies several high-impact neurological diseases including multiple sclerosis (MS), stroke, and Alzheimer's disease. Neuroinflammation is sterile, as damage-associated molecular patterns rather than microbial pathogens elicit the response. The inflammasome, which leads to caspase-1 activation, is implicated in neuroinflammation. In this study, we reveal that lysophosphatidylcholine (LPC), a molecule associated with neurodegeneration and demyelination, elicits NLRP3 and NLRC4 inflammasome activation in microglia and astrocytes, which are central players in neuroinflammation. LPC-activated inflammasome also requires ASC (apoptotic speck containing protein with a CARD), caspase-1, cathepsin-mediated degradation, calcium mobilization, and potassium efflux but not caspase-11. To study the physiological relevance, Nlrc4-/- and Nlrp3-/- mice are studied in the cuprizone model of neuroinflammation and demyelination. Mice lacking both genes show the most pronounced reduction in astrogliosis and microglial accumulation accompanied by decreased expression of the LPC receptor G2A, whereas MS patient samples show increased G2A. These results reveal that NLRC4 and NLRP3, which normally form distinct inflammasomes, activate an LPC-induced inflammasome and are important in astrogliosis and microgliosis.

GLT-1-Dependent Disruption of CNS Glutamate Homeostasis and Neuronal Function by the Protozoan Parasite Toxoplasma gondii.

The immune privileged nature of the CNS can make it vulnerable to chronic and latent infections. Little is known about the effects of lifelong brain infections, and thus inflammation, on the neurological health of the host. Toxoplasma gondii is a parasite that can infect any mammalian nucleated cell with average worldwide seroprevalence rates of 30%. Infection by Toxoplasma is characterized by the lifelong presence of parasitic cysts within neurons in the brain, requiring a competent immune system to prevent parasite reactivation and encephalitis. In the immunocompetent individual, Toxoplasma infection is largely asymptomatic, however many recent studies suggest a strong correlation with certain neurodegenerative and psychiatric disorders. Here, we demonstrate a significant reduction in the primary astrocytic glutamate transporter, GLT-1, following infection with Toxoplasma. Using microdialysis of the murine frontal cortex over the course of infection, a significant increase in extracellular concentrations of glutamate is observed. Consistent with glutamate dysregulation, analysis of neurons reveal changes in morphology including a reduction in dendritic spines, VGlut1 and NeuN immunoreactivity. Furthermore, behavioral testing and EEG recordings point to significant changes in neuronal output. Finally, these changes in neuronal connectivity are dependent on infection-induced downregulation of GLT-1 as treatment with the ß-lactam antibiotic ceftriaxone, rescues extracellular glutamate concentrations, neuronal pathology and function. Altogether, these data demonstrate that following an infection with T. gondii, the delicate regulation of glutamate by astrocytes is disrupted and accounts for a range of deficits observed in chronic infection.

Antitumor activity of a polypyridyl chelating ligand: in vitro and in vivo inhibition of glioma.

Glioblastoma multiforme is an extremely aggressive and invasive form of central nervous system tumor commonly treated with the chemotherapeutic drug Temozolomide. Unfortunately, even with treatment, the median survival time is less than 12 months. 2,9-Di-sec-butyl-1,10-phenanthroline (SBP), a phenanthroline-based ligand originally developed to deliver gold-based anticancer drugs, has recently been shown to have significant antitumor activity in its own right. SBP is hypothesized to initiate tumor cell death via interaction with non-DNA targets, and considering most glioblastoma drugs kill tumors through DNA damage processes, SBP was tested as a potential novel drug candidate against glial-based tumors. In vitro studies demonstrated that SBP significantly inhibited the growth of rodent GL-26 and C6 glioma cells, as well as human U-87, and SW1088 glioblastomas/astrocytomas. Furthermore, using a syngeneic glioma model in mice, in vivo administration of SBP significantly reduced tumor volume and increased survival time. There was no significant toxicity toward nontumorigenic primary murine and human astrocytes in vitro, and limited toxicity was observed in ex vivo tissues obtained from noncancerous mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and recovery assays suggest that SBP induces apoptosis in gliomas. This exploratory study suggests SBP is effective in slowing the growth of tumorigenic cells in the brain while exhibiting limited toxicity to normal cells and tissues and should therefore be further investigated for its potential in glioblastoma treatment.

Selective cavitand-mediated endocytosis of targeted imaging agents into live cells.

A water-soluble synthetic receptor molecule is capable of selective, controlled endocytosis of a specifically tagged target molecule in different types of living human cells. The presence of suitable choline-derived binding handles is essential for the molecular recognition and transport process, allowing selective guest transport and imaging of cancer cells.

Chitinase dependent control of protozoan cyst burden in the brain.

Chronic infections represent a continuous battle between the host's immune system and pathogen replication. Many protozoan parasites have evolved a cyst lifecycle stage that provides it with increased protection from environmental degradation as well as endogenous host mechanisms of attack. In the case of Toxoplasma gondii, these cysts are predominantly found in the immune protected brain making clearance of the parasite more difficult and resulting in a lifelong infection. Currently, little is known about the nature of the immune response stimulated by the presence of these cysts or how they are able to propagate. Here we establish a novel chitinase-dependent mechanism of cyst control in the infected brain. Despite a dominant Th1 immune response during Toxoplasma infection there exists a population of alternatively activated macrophages (AAMØ) in the infected CNS. These cells are capable of cyst lysis via the production of AMCase as revealed by live imaging, and this chitinase is necessary for protective immunity within the CNS. These data demonstrate chitinase activity in the brain in response to a protozoan pathogen and provide a novel mechanism to facilitate cyst clearance during chronic infections.

Stabilin-1 expression in tumor associated macrophages.

Glioblastoma multiforme is a very aggressive and common form of brain tumor. Current therapies consist of a combination of surgical removal, chemotherapy and radiation therapy. These drastic treatments still leave a current prognosis of median survival of less than 1 year. Lack of effectiveness of these treatments has left researchers looking for alternative forms of treatment. A significant alternative currently being investigated is the use of the immune system to potentially target and eliminate tumor cells directly. Stabilin-1, a scavenger receptor expressed by macrophages, has the potential in inhibiting tumor growth by binding and internalizing secreted protein acidic and rich in cysteine (SPARC). SPARC is known to be upregulated in the tumor microenvironment and is involved in extracellular matrix remodeling, cell proliferation and migration. Decreasing SPARC expression using siRNA has been shown to decrease tumor invasiveness and survival. We investigated the phenotype of stabilin-1 expressing immune cells in the tumor environment and demonstrated a transient population of alternatively activated macrophages expressing stabilin-1 in the tumor environment and the disappearance of that population as the tumor progresses.