NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.

Staphylococcus aureus AbcA transporter enhances persister formation under ß-lactam exposure.

We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6-14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

Systemic Immune Bias Delineates Malignant Astrocytoma Survival Cohorts.

Patients with grade III anaplastic astrocytomas (AA) separate into survival cohorts based on the presence or absence of mutations in isocitrate dehydrogenase (IDH). Progression to glioblastoma (GBM), morphologically distinguishable by elevated microvascular proliferation, necrosis, and cell division in tumor tissues, is considerably more rapid in IDH wild-type tumors such that their diagnosis as AA is relatively rare. More often initially presenting as GBM, these contain higher numbers of tumor-associated macrophages (TAMs) than most AA, and GBM patients also have higher levels of circulating M2 monocytes. TAM and M2 monocytes share functional properties inhibitory for antitumor immunity. Yet, although there is a wealth of data implicating TAM in tumor-immune evasion, there has been limited analysis of the impact of the circulating M2 monocytes. In the current study, immune parameters in sera, circulating cells, and tumor tissues from patients with primary gliomas morphologically diagnosed as AA were assessed. Profound differences in serum cytokines, glioma extracellular vesicle cross-reactive Abs, and gene expression by circulating cells identified two distinct patient cohorts. Evidence of type 2-immune bias was most often seen in patients with IDH wild-type AA, whereas a type 1 bias was common in patients with tumors expressing the IDH1R132H mutation. Nevertheless, a patient's immune profile was better correlated with the extent of tumor vascular enhancement on magnetic resonance imaging than IDH mutational status. Regardless of IDH genotype, AA progression appears to be associated with a switch in systemic immune bias from type 1 to type 2 and the loss of tumor vasculature integrity.

Role of Staphylococcus aureus Tet38 in Transport of Tetracycline and Its Regulation in a Salt Stress Environment.

Staphylococcus aureus Tet38 efflux pump has multiple functions, including conferring resistance to tetracycline and other compounds and enabling internalization and survival within epithelial cells. In this study, we evaluated the effects of sodium and potassium on tet38 expression. These monovalent cations are known to play a role in transport by the related S. aureus TetK and B. subtilis TetL transporters. tet38 transcription decreased with increasing sodium concentrations by means of direct repression by the salt stress-dependent KdpD/E regulator. tet38 transcription increased 20-fold and tetracycline minimum inhibitory concentration (MIC) increased 4-fold in a ΔkdpD mutant. KdpE bound specifically to the tet38 promoter. Under extreme salt stress, the survival of S. aureus with intact tet38 was reduced compared to that of a Δtet38 mutant. To study the effect of sodium on Tet38 function, we generated constructs overexpressing tet38 and tetK and introduced them into Escherichia coli TO114, which is deficient in major sodium transporters. Tet38 tetracycline efflux was directly demonstrated in a fluorescence assay, and tetracycline efflux of both Tet38 and TetK was abolished by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In contrast, NaCl inhibited efflux by Tet38 but not TetK, whereas KCl inhibited efflux by TetK but not Tet38. Cell-associated Na increased with heterologous overexpression of Tet38. These data indicate that S. aureus Tet38 is a tetracycline efflux pump regulated by the KdpD/E regulator. Under salt stress, S. aureus adjusted its survival in part by reducing the expression of tet38 through KdpD/E. The mechanisms by which Tet38 is detrimental to salt tolerance in S. aureus and inhibited by sodium remain to be determined. IMPORTANCE This study shows that S. aureus Tet38 is a tetracycline efflux pump regulated by KdpD/E regulator. These findings are the first direct demonstration of Tet38-mediated tetracycline efflux, which had previously been inferred from its ability to confer tetracycline resistance. Under salt stress, S. aureus adjusts its survival in part by reducing the expression of tet38 through KdpD/E. We demonstrated the differences in the respective functions of S. aureus Tet38 and other tetracycline efflux transporters (S. aureus TetK, B. subtilis TetL) regarding their transport of tetracycline and Na+/K+. Notably, sodium selectively reduced tetracycline efflux by Tet38, and potassium selectively reduced tetracycline efflux by TetK. The multiple functions of Tet38 emphasize its importance in bacterial adaptation to and survival in diverse environments.

Staphylococcus aureus Efflux Pumps and Tolerance to Ciprofloxacin and Chlorhexidine following Induction by Mupirocin.

Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a yellow fluorescent protein (YFP) fluorescence reporter of NorA. Mupirocin exposure also produced reduced susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact norA and mepA, respectively.

Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization.

The Staphylococcus aureus Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F411P412G413, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D38D39L40, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R106 and G151 (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.

Dual Role of Toll-Like Receptor 7 in the Pathogenesis of Rabies Virus in a Mouse Model.

Rabies, caused by rabies virus (RABV), is a fatal encephalitis in humans and other mammals, which continues to present a public health threat in most parts of the world. Our previous study demonstrated that Toll-like receptor 7 (TLR7) is essential in the induction of anti-RABV antibodies via the facilitation of germinal center formation. In the present study, we investigated the role of TLR7 in the pathogenicity of RABV in a mouse model. Using isolated plasmacytoid dendritic cells (pDCs), we demonstrated that TLR7 is an innate recognition receptor for RABV. When RABV invaded from the periphery, TLR7 detected viral single-stranded RNA and triggered immune responses that limited the virus's entry into the central nervous system (CNS). When RABV had invaded the CNS, its detection by TLR7 led to the production of cytokines and chemokines and an increase the permeability of the blood-brain barrier. Consequently, peripheral immune cells, including pDCs, macrophages, neutrophils, and B cells infiltrated the CNS. While this immune response, triggered by TLR7, helped to clear viruses, it also increased neuroinflammation and caused immunopathology in the mouse brain. Our results demonstrate that TLR7 is an innate recognition receptor for RABV, which restricts RABV invasion into the CNS in the early stage of viral infection but also contributes to immunopathology by inducing neuroinflammation.IMPORTANCE Developing targeted treatment for RABV requires understanding the innate immune response to the virus because early virus clearance is essential for preventing the fatality when the infection has progressed to the CNS. Previous studies have revealed that TLR7 is involved in the immune response to RABV. Here, we establish that TLR7 recognizes RABV and facilitates the production of some interferon-stimulated genes. We also demonstrated that when RABV invades into the CNS, TLR7 enhances the production of inflammatory cytokines which contribute to immunopathology in the mouse brain. Taken together, our findings suggest that treatments for RABV must consider the balance between the beneficial and harmful effects of TLR7-triggered immune responses.

Cosmic-ray antinuclei as messengers of new physics: status and outlook for the new decade.

The precise measurement of cosmic-ray antinuclei serves as an important means for identifying the nature of dark matter and other new astrophysical phenomena, and could be used with other cosmic-ray species to understand cosmic-ray production and propagation in the Galaxy. For instance, low-energy antideuterons would provide a "smoking gun" signature of dark matter annihilation or decay, essentially free of astrophysical background. Studies in recent years have emphasized that models for cosmic-ray antideuterons must be considered together with the abundant cosmic antiprotons and any potential observation of antihelium. Therefore, a second dedicated Antideuteron Workshop was organized at UCLA in March 2019, bringing together a community of theorists and experimentalists to review the status of current observations of cosmic-ray antinuclei, the theoretical work towards understanding these signatures, and the potential of upcoming measurements to illuminate ongoing controversies. This review aims to synthesize this recent work and present implications for the upcoming decade of antinuclei observations and searches. This includes discussion of a possible dark matter signature in the AMS-02 antiproton spectrum, the most recent limits from BESS Polar-II on the cosmic antideuteron flux, and reports of candidate antihelium events by AMS-02; recent collider and cosmic-ray measurements relevant for antinuclei production models; the state of cosmic-ray transport models in light of AMS-02 and Voyager data; and the prospects for upcoming experiments, such as GAPS. This provides a roadmap for progress on cosmic antinuclei signatures of dark matter in the coming years.

Tet38 of Staphylococcus aureus Binds to Host Cell Receptor Complex CD36-Toll-Like Receptor 2 and Protects from Teichoic Acid Synthesis Inhibitors Tunicamycin and Congo Red.

Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of Staphylococcus aureus bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not to TLR-2 alone or TLR-2 and S. aureus lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of S. aureustet38 mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of tet38 and a 2-fold increase in activity in QT7 (tet38). Reserpine (an inhibitor of efflux pumps) reduced the effect of tet38 overexpression on tunicamycin resistance 4-fold. In addition, tet38 affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of tet38 reducing growth. In conclusion, Tet38 contributes to S. aureus invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the tet38 overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in S. aureus against their inhibitors, possibly functioning as an efflux pump.

Type 1 Immune Mechanisms Driven by the Response to Infection with Attenuated Rabies Virus Result in Changes in the Immune Bias of the Tumor Microenvironment and Necrosis of Mouse GL261 Brain Tumors.

Immunotherapeutic strategies for malignant glioma have to overcome the immunomodulatory activities of M2 monocytes that appear in the circulation and as tumor-associated macrophages (TAMs). M2 cell products contribute to the growth-promoting attributes of the tumor microenvironment (TME) and bias immunity toward type 2, away from the type 1 mechanisms with antitumor properties. To drive type 1 immunity in CNS tissues, we infected GL261 tumor-bearing mice with attenuated rabies virus (RABV). These neurotropic viruses spread to CNS tissues trans-axonally, where they induce a strong type 1 immune response that involves Th1, CD8, and B cell entry across the blood-brain barrier and virus clearance in the absence of overt sequelae. Intranasal infection with attenuated RABV prolonged the survival of mice bearing established GL261 brain tumors. Despite the failure of virus spread to the tumor, infection resulted in significantly enhanced tumor necrosis, extensive CD4 T cell accumulation, and high levels of the proinflammatory factors IFN-γ, TNF-α, and inducible NO synthase in the TME merely 4 d postinfection, before significant virus spread or the appearance of RABV-specific immune mechanisms in CNS tissues. Although the majority of infiltrating CD4 cells appeared functionally inactive, the proinflammatory changes in the TME later resulted in the loss of accumulating M2 and increased M1 TAMs. Mice deficient in the Th1 transcription factor T-bet did not gain any survival advantage from RABV infection, exhibiting only limited tumor necrosis and no change in TME cytokines or TAM phenotype and highlighting the importance of type 1 mechanisms in this process.

Tet38 Efflux Pump Contributes to Fosfomycin Resistance in Staphylococcus aureus.

Fosfomycin inhibits MurA following uptake by the GlpT transporter of glycerol-3-phosphate in Escherichia coli In Staphylococcus aureus, plasmid overexpression of the Tet38 efflux pump and a glpT mutant resulted in increased MICs and decreased accumulation of fosfomycin, with MICs affected by glycerol-3-phosphate. In contrast, a tet38 mutant had a lower MIC and increased accumulation of fosfomycin, suggesting that Tet38 acts as an efflux transporter of fosfomycin.

Clearance of attenuated rabies virus from brain tissues is required for long-term protection against CNS challenge with a pathogenic variant.

Rabies virus is a neurotropic lyssavirus which is 100% fatal in its pathogenic form when reaching unprotected CNS tissues. Death can be prevented by mechanisms delivering appropriate immune effectors across the blood-brain barrier which normally remains intact during pathogenic rabies virus infection. One therapeutic approach is to superinfect CNS tissues with attenuated rabies virus which induces blood-brain barrier permeability and immune cell entry. Current thinking is that peripheral rabies immunization is sufficient to protect against a challenge with pathogenic rabies virus. While this is undoubtedly the case if the virus is confined to the periphery, what happens if the virus reaches the CNS is less well-understood. In the current study, we find that peripheral immunization does not fully protect mice long-term against an intranasal challenge with pathogenic rabies virus. Protection is significantly better in mice that have cleared attenuated virus from the CNS and is associated with a more robust CNS recall response evidently due to the presence in CNS tissues of elevated numbers of lymphocytes phenotypically resembling long-term resident immune cells. Adoptive transfer of cells from rabies-immune mice fails to protect against CNS challenge with pathogenic rabies virus further supporting the concept that long-term resident immune cell populations must be established in brain tissues to protect against a subsequent CNS challenge with pathogenic rabies virus.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes.

Transcriptional Regulator TetR21 Controls the Expression of the Staphylococcus aureus LmrS Efflux Pump.

TetR21 controls the expression of Tet38 and LmrS efflux pumps. A tetR21 mutant, QT21, exhibited a 4-fold increase in the transcription level of lmrSStaphylococcus aureuslmrS overexpressor showed increases of 4-fold and 2-fold, respectively, in the MICs of chloramphenicol and erythromycin, while the MICs of lmrS mutant QT18 and lmrS-tetR21 mutant QT1821 remained similar to those of parental strain RN6390. TetR21 does not bind to the promoter of lmrS, suggesting indirect regulation of lmrS.

T-bet Is Required for the Rapid Clearance of Attenuated Rabies Virus from Central Nervous System Tissue.

Much of our understanding of CNS immunity has been gained from models involving pathological inflammation. Attenuated rabies viruses (RABV) are unique tools to study CNS immunity in the absence of conventional inflammatory mechanisms, as they spread from the site of inoculation to the CNS transaxonally, thereby bypassing the blood-brain barrier (BBB), and are cleared without neutrophil or monocyte infiltration. To better understand the role of CD4 T cell subsets in the clearance of the virus from CNS tissues, we examined the development of antiviral immunity in wild-type (WT) and T-bet knockout mice (T-bet(-/-)), which lack Th1 cells. Early control of RABV replication in the CNS tissues of WT mice is associated with the production of IFN-γ, with antiviral effects likely mediated through the enhanced expression of type I IFNs. Of interest, IFN-α and -γ are overexpressed in the infected T-bet(-/-) by comparison with WT CNS tissues, and the initial control of RABV infection is similar. Ultimately, attenuated RABV are cleared from the CNS tissues of WT mice by Ab locally produced by the activities of infiltrating T and B cells. Although T and B cell infiltration into the CNS of infected T-bet(-/-) mice is comparable, their activities are not, the consequence being delayed, low-level Ab production and prolonged RABV replication. More importantly, neither T-bet(-/-) mice immunized with an attenuated virus, nor WT mice with Th2 RABV-specific immunity induced by immunization with inactivated virus, are protected in the long term against challenge with a pathogenic RABV.

Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon.

UNLABELLED: Previous animal model experiments have shown a correlation between interferon gamma (IFN-γ) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-γ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-γ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBNγ. Morbidity and mortality were monitored in mice infected intranasally with SPBNγ or SPBN(-) control virus to determine the degree of attenuation caused by the expression of IFN-γ. Incorporation of IFN-γ into the rabies virus genome highly attenuated the virus. SPBNγ has a 50% lethal dose (LD50) more than 100-fold greater than SPBN(-). In vitro and in vivo mouse experiments show that SPBNγ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR(-/-)) cannot control the SPBNγ infection and rapidly die. These data suggest that IFN-γ production has antiviral effects in rabies, largely due to the induction of type I interferons. IMPORTANCE: Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-γ production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-γ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-γ directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN-γ to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.

Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells.

We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.

Clinical Practice of Steroid Avoidance in Pediatric Kidney Transplantation.

Steroid-avoidance protocols have recently gained popularity in pediatric kidney transplantation. We investigated the clinical practice of steroid avoidance among 9494 kidney transplant recipients at 124 transplant centers between 2000 and 2012 in the Organ Procurement and Transplantation Network database. The practice of steroid avoidance increased during the study period and demonstrated significant variability among transplant centers. From 2008 to 2012, 39% of transplant centers used steroid avoidance in <10% of all discharged transplant recipients. Twenty-one percent of transplant centers practiced steroid avoidance in 10-40% of transplant recipients, and 40% of transplant centers used steroid avoidance in >40% of discharged patients. Children receiving steroid avoidance more frequently received induction with lymphocyte-depleting agents. Repeat kidney transplants were the least likely to receive steroid avoidance. Children who received a deceased donor kidney, underwent pretransplant dialysis, were highly sensitized, or had glomerular kidney disease or delayed graft function were also less likely to receive steroid avoidance. The variation in practice between centers remained highly significant (p < 0.0001) after adjustment for all patient- and center-level factors in multivariate analysis. We conclude that significant differences in the practice of steroid avoidance among transplant centers remain unexplained and may reflect uncertainty about the safety and efficacy of steroid-avoidance protocols.

Glioblastoma exosomes and IGF-1R/AS-ODN are immunogenic stimuli in a translational research immunotherapy paradigm.

Glioblastomas are primary intracranial tumors for which there is no cure. Patients receiving standard of care, chemotherapy and irradiation, survive approximately 15 months prompting studies of alternative therapies including vaccination. In a pilot study, a vaccine consisting of Lucite diffusion chambers containing irradiated autologous tumor cells pre-treated with an antisense oligodeoxynucleotide (AS-ODN) directed against the insulin-like growth factor type 1 receptor was found to elicit positive clinical responses in 8/12 patients when implanted in the rectus sheath for 24 h. Our preliminary observations supported an immune response, and we have since reopened a second Phase 1 trial to assess this possibility among other exploratory objectives. The current study makes use of a murine glioma model and samples from glioblastoma patients in this second Phase 1 trial to investigate this novel therapeutic intervention more thoroughly. Implantation of the chamber-based vaccine protected mice from tumor challenge, and we posit this occurred through the release of immunostimulatory AS-ODN and antigen-bearing exosomes. Exosomes secreted by glioblastoma cultures are immunogenic, eliciting and binding antibodies present in the sera of immunized mice. Similarly, exosomes released by human glioblastoma cells bear antigens recognized by the sera of 6/12 patients with recurrent glioblastomas. These results suggest that the release of AS-ODN together with selective release of exosomes from glioblastoma cells implanted in chambers may drive the therapeutic effect seen in the pilot vaccine trial.

Enhancement of glioma-specific immunity in mice by "NOBEL", an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide.

Autologous glioblastoma multiforme tumor cells treated with an antisense oligodeoxynucleotide (AS-ODN) targeting insulin-like growth factor receptor-1 (IGF-1R) are the basis of a vaccine with therapeutic effects on tumor recurrence in a pilot clinical trial. As a preface to continued clinical investigation of this vaccination strategy, we have studied the contribution of an optimized IGF-1R AS-ODN, designated "NOBEL", to the induction of immunity to mouse GL261 glioma cells. The impact of NOBEL on mechanisms contributing to the development of GL261 immunity was first examined in the periphery. GL261 cells are naturally immunogenic when implanted into the flanks of congenic C57BL/6 mice, immunizing rather than forming tumors in around 50 % of these animals but causing tumors in the majority of mice lacking T and B lymphocytes. Overnight treatment with NOBEL in vitro reduces IGF-1R expression by GL261 cells but has minimal effect on cell viability and does not reduce the capacity of the cells to form tumors upon implantation. In contrast, tumors are extremely rare when GL261 cells are mixed with NOBEL at inoculation into the flanks of C57BL/6, and the recipient mice become immune to subcutaneous and intracranial challenge with untreated GL261. Adaptive immune mechanisms contribute to this effect, as immunocompromised mice fail to either fully control tumor formation or develop immunity following flank administration of the GL261/NOBEL mix. NOBEL's structure has known immunostimulatory motifs that likely contribute to the immunogenicity of the mix, but its specificity for IGF-1R mRNA is also important as a similarly structured sense molecule is not effective.