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Stroke family caregivers often neglect their own health while providing care. Rigorous reviews have focused on stroke caregiver needs and outcomes; however, a comprehensive review of stroke caregiver health is lacking. The purpose of this integrative review was to determine factors associated with stroke family caregiver health. Using a PRISMA flow diagram and Rayyan software, 41 studies were identified published from January 2000 to December 2020. Databases included Cochrane Reviews, Cochrane Trials, PsycINFO, Ovid MEDLINE, PubMed, EBSCOhost MEDLINE, Embase, and CINAHL. Rigorous guidelines were used to critique the 41 articles. Health measures were global in nature, lacking details regarding health promotion activities important to stroke family caregiver health. Common factors associated with caregiver health were depressive symptoms and burden. Further research is needed to design more situation-specific instruments to measure stroke family caregiver health, as well as interventions to reduce depressive symptoms and burden while promoting caregiver health.

Purpose: Despite standard of care (SOC) established by Stupp, glioblastoma remains a uniformly poor prognosis. We evaluated IGV-001, which combines autologous glioblastoma tumor cells and an antisense oligonucleotide against IGF type 1 receptor (IMV-001), in newly diagnosed glioblastoma. Patients and Methods: This open-label protocol was approved by the Institutional Review Board at Thomas Jefferson University. Tumor cells collected during resection were treated ex vivo with IMV-001, encapsulated in biodiffusion chambers with additional IMV-001, irradiated, then implanted in abdominal acceptor sites. Patients were randomized to four exposure levels, and SOC was initiated 4–6 weeks later. On the basis of clinical improvements, randomization was halted after patient 23, and subsequent patients received only the highest exposure. Safety and tumor progression were primary and secondary objectives, respectively. Time-to-event outcomes were compared with the SOC arms of published studies. Results: Thirty-three patients were enrolled, and median follow-up was 3.1 years. Six patients had adverse events (grade ≤3) possibly related to IGV-001. Median progression-free survival (PFS) was 9.8 months in the intent-to-treat population (vs. SOC, 6.5 months; P = 0.0003). In IGV-001–treated patients who met Stupp-eligible criteria, PFS was 11.6 months overall (n = 22; P = 0.001) and 17.1 months at the highest exposure (n = 10; P = 0.0025). The greatest overall survival was observed in Stupp-eligible patients receiving the highest exposure (median, 38.2 months; P = 0.044). Stupp-eligible patients with methylated O6-methylguanine–DNA methyltransferase promoter (n = 10) demonstrated median PFS of 38.4 months (P = 0.0008). Evidence of immune activation was noted. Conclusions: IGV-001 was well tolerated, PFS compared favorably with SOC, and evidence suggested an immune-mediated mechanism (ClinicalTrials.gov: NCT02507583).

Immune memory cells residing in previously infected, nonlymphoid tissues play a role in immune surveillance. In the event that circulating antibodies fail to prevent virus spread to the tissues in a secondary infection, these memory cells provide an essential defense against tissue reinfection. CNS tissues are isolated from circulating immune cells and antibodies by the blood–brain barrier, making the presence of tissue-resident immune memory cells particularly needed to combat recurrent infection by neurotropic viruses. Wild-type and laboratory-engineered rabies viruses are neurotropic, differ in pathogenicity, and have varying effects on BBB functions. These viruses have proven invaluable tools in demonstrating the importance of tissue-resident immune memory cells in the reinfection of CNS tissues. Only Type 1 immune memory is effective at therapeutically clearing a secondary infection with wild-type rabies viruses from the CNS and does so despite the maintenance of blood–brain barrier integrity.

The threat of workplace violence (WPV) is a significant occupational hazard for home healthcare workers (HHCWs). The purpose of this integrative review is to examine WPV interventions used by HHCWs to stay safe while working in the patient's home and community. The methodology used was the integrative review by , which allows for inclusion of experimental and non-experimental research, reflecting the state of the science on interventions used by HHCWs to mitigate and prevent WPV. A total of 17 articles pertained to interventions used by HHCWs. Interventions were further categorized by WPV Type. There are a number of interventions used for Type I and II WPV. However, interventions for Type III WPV are minimal and interventions for Type IV WPV are obsolete. Safety and health training were shown to be significant in increasing HHCWs' confidence and knowledge about WPV prevention. Researchers demonstrated safety and health training are effective in promoting a safe work environment and reducing incidents of WPV. This review begins to fill the gap in the literature on interventions used by HHCWs to mitigate and prevent WPV.

The purpose of this systematic review is to describe the operationalization of interruptions measurement and to synthesize the evidence on the causes and consequences of interruptions in the emergency department (ED) work environment.This systematic review of studies explores the causes and consequences of interruptions in the ED. Of 2836 abstract/titles screened, 137 full-text articles were reviewed, and 44 articles met inclusion criteria of measuring ED interruptions.All articles reported primary data collection, and most were cohort studies (n = 30, 68%). Conceptual or operational definitions of interruptions were included in 27 articles. Direct observation was the most common approach. In half of the studies, quantitative measures of interruptions in the ED were descriptive only, without measurements of interruptions' consequences. Twenty-two studies evaluated consequences, including workload, delays, satisfaction, and errors. Overall, relationships between ED interruptions and their causes and consequences are primarily derived from direct observation within large academic hospitals using heterogeneous definitions. Collective strengths of interruptions research in the ED include structured methods of naturalistic observation and definitions of interruptions derived from concept analysis. Limitations are conflicting and complex evaluations of consequences attributed to interruptions, including the predominance of descriptive reports characterizing interruptions without direct measurements of consequences.The use of standardized definitions and measurements in interruptions research could contribute to measuring the impact and influence of interruptions on clinicians' productivity and efficiency as well as patients' outcomes, and thus provide a basis for intervention research.

Objectives There is no study published regarding the benefit of radiation therapy (RT) in combination with immune checkpoint inhibitors (ICIs) for the treatment of metastatic renal cell cancer (mRCC). This report is part of an exploratory study aiming to determine the immunomodulatory activity of RT alone or in combination with pembrolizumab in solid tumors. Materials and methods mRCC patients were treated with a combination of RT (8 Gy×1 or 4 Gy×5) followed by pembrolizumab with or without lead-in dose of pembrolizumab. Treatment response was measured based on the modified Response Evaluation Criteria in Solid Tumors criteria. Adverse events were monitored and graded. Pre-RT and post-RT tumor biopsies were obtained to evaluate programmed death-ligand 1 expression. Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry. Results Twelve mRCC patients who progressed on prior antiangiogenic therapy were enrolled. Half had 2 lines of prior therapy. Two patients (16.7%) had partial responses and were on study for 12.4 and 14.5 months. Three patients had stable disease for a period ranging from 4.2 to 10.4 months, whereas 7 patients had progressive disease. Median progression-free survival was 8.6 months and median overall survival was 32.3 months. Three patients had grade ≥3 events (hyperglycemia, thrombocytopenia, transaminitis). Biopsied tissue programmed death-ligand 1 expression and tumor-infiltrating lymphocytes were numerically higher in responders comparing to nonresponders (Modified Proportion Score 45% vs. 30.45%; tumor-infiltrating lymphocytes odds ratio 4.92). Conclusion Combining RT with pembrolizumab in pretreated mRCC is well-tolerated and appears to have comparable efficacy with single-agent nivolumab.

Scintigraphic imaging of malignant glioblastoma (MG) continues to be challenging. We hypothesized that VPAC1 cell surface receptors can be targeted for positron emission tomography (PET) imaging of orthotopically implanted MG in a mouse model, using a VPAC1-specific peptide [64Cu]TP3805. The expression of VPAC1 in mouse GL261 and human U87 glioma cell lines was determined by western blot. The ability of [64Cu]TP3805 to bind to GL261 and U87 cells was studied by cell-binding. Receptor-blocking studies were performed to validate receptor specificity. GL261 tumors were implanted orthotopically in syngeneic T-bet knockout C57BL/6 mouse brain (N = 15) and allowed to grow for 2–3 weeks. Mice were injected i.v., first with ~ 150 μCi of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) then 24 h later with ~ 200 μCi of [64Cu]TP3805. In another set of tumor-bearing mice, (N = 5), ionic [64Cu]Cl2 was injected as a control. Mice were imaged at a 2-h post-injection using an Inveon micro-PET/CT, sacrificed and % ID/g of [64Cu]TP3805 and [64Cu]Cl2 were calculated in a tumor, normal brain, and other tissues. For histologic tissue examination, 3-μm thick sections of the tumors and normal brain were prepared, digital autoradiography (DAR) was performed, and then the sections were H&E stained for histologic examination. Western blots showed a strong signal for VPAC1 on both cell lines. [64Cu]TP3805 cell-binding was 87 ± 1.5 %. Receptor-blocking reduced cell-binding to 24.3 ± 1.5 % (P

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.

Previous studies have suggested that maternal supplementation with n-3 long-chain polyunsaturated fatty acids may reduce the incidence of preterm delivery but may also prolong gestation beyond term; however, more data are needed regarding the role of n-3 long-chain polyunsaturated fatty acids in pregnancy.We performed a multicenter, double-blind, randomized trial in which women who were pregnant with single or multiple fetuses were assigned to receive either fish-oil capsules that contained 900 mg of n-3 long-chain polyunsaturated fatty acids (n-3 group) or vegetable-oil capsules that contained trace n-3 long-chain polyunsaturated fatty acids (control group) daily, beginning before 20 weeks of gestation and continuing to 34 weeks of gestation or delivery, whichever occurred first. The primary outcome was early preterm delivery, defined as delivery before 34 completed weeks of gestation. Other pregnancy and neonatal outcomes were also assessed.A total of 5544 pregnancies in 5517 women were randomly assigned at six centers in Australia; 5486 pregnancies were included in the primary analysis. Early preterm delivery occurred in the case of 61 of 2734 pregnancies (2.2%) in the n-3 group and 55 of 2752 pregnancies (2.0%) in the control group; the between-group difference was not significant (adjusted relative risk, 1.13; 95% confidence interval [CI], 0.79 to 1.63; P = 0.50). There were no significant differences between the groups in the incidence of interventions in post-term (41 weeks of gestation) deliveries, in adverse events, or in other pregnancy or neonatal outcomes, except that a higher percentage of infants born to women in the n-3 group than in the control group were very large for gestational age at birth (adjusted relative risk, 1.30; 95% CI, 1.02 to 1.65). Percentages of serious adverse events did not differ between the groups. Minor gastrointestinal disturbances were more commonly reported in the n-3 group than in the control group.Supplementation with n-3 long-chain polyunsaturated fatty acids from early pregnancy (20 weeks of gestation) until 34 weeks of gestation did not result in a lower incidence of early preterm delivery or a higher incidence of interventions in post-term deliveries than control. (Funded by the Australian National Health and Medical Research Council and the Thyne Reid Foundation; ORIP Australian New Zealand Clinical Trials Registry number, ACTRN12613001142729.).

Introduction: We present a novel autologous cell vaccine therapy designed to treat patients with newly diagnosed glioblastoma (Trial Registration: IND 14379, NCT01550523). Methods: This phase 1b trial has a phase 2 design with 4 randomized vaccine dose cohorts in 33 patients with the objective being safety assessment but also including clinical, radiographic, and immune analyses. Eligibility criteria included age > 18 and Karnofsky score of > 60; neither bihemispheric disease nor extent of resection were exclusion criteria but autoimmune diseases were. During craniotomy for tumor resection, if frozen section confirmed GBM, incisions were made in the lower abdomen through the rectus sheath and pockets created between the sheath and the muscle and the wounds closed with a temporary three-layer closure. Tumor resection involved an aspirator that collected morselized viable tumor tissue in sterile traps. The tissue was processed by overnight culture with 0.2 mg of an IGF-1R antisense oligodeoxynucleotide/gm. The next (first postoperative) day, treated tumor cells were harvested, encapsulated in either ten or twenty small biodiffusion chambers along with 4 micrograms of the IGF-1R antisense, irradiated and then implanted at bedside in the abdominal acceptor sites as previously described (1). Chambers were explanted 24 or 48 hours later, depending on randomization. Standard of care according to Stupp (2) was initiated at 6 weeks. Studies included 3T MRI imaging and analysis of serial blood samples for T cell function and cytokine levels. Disease progression was assessed using RANO (3) and iRANO (4) criteria with a data cutoff of March 1 (N=30) used for this analysis. Results: The trial opened September 1, 2015 and completed accrual on March 1, 2018. A midpoint interim analysis revealed significantly more robust cytokine responses at the highest vaccine dose. Randomization was therefore stopped at subject 23 and amended to treat using only the highest dose. Progression-free survival (PFS) was compared to three historic SOC comparators (Stupp [2], Kong [5], and an antecedent cohort of 37 consecutive patients treated with SOC at our institution [TJUH]). PFS was significantly improved at 10.5 mo v. SOC comparators: 6.9 mo (Stupp, p = .003), 5.3 mo (Kong, p = .002) and 7 mo (TJUH, p = .013). Seventy-five percent of the 14 patients in the highest-dose cohort had robust proinflammatory and early evidence of sustained immune reactivity associated with tumor regression or no recurrence after surgery. Conclusion: These data reflect a therapeutic benefit defined as significant improvement in PFS without increased safety risk compared to three different SOC cohorts. Since GBM remains one of the most challenging solid tumors, this treatment design invites investigator collaboration in a multicenter phase 2 trial. References: 1. Andrew et al. J Clin Oncol 19:2189-2200; 2. Stupp et al. NEJM 352:987-96; 3. Wen et al. J Clin. Oncol 28:1963-72; 4. Okada et al. Lancet Oncol 16:534-42; 5. Kong et al. Oncotarget 8:7003-13. Citation Format: Kevin D. Judy, David W. Andrews, Larry Harshyne, Lawrence Kenyon, Kiran Talekar, Kofi-Buaku Atsina, Lyndon Kim, Wenyin Shi, Maria Werner-Wasik, Rhonda Kean, Samantha Garcia, Kara Pigott, Charles B. Scott, D. Craig Hooper. Phase 1b/2 prospective randomized trial of four autologous cell vaccine dose cohorts for initial treatment of glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B71.

We describe here a novel process for the production by glioma cells of cell-free, tumor-specific antigens that supplant the need for intact tumor cells in the induction of immunity against autologous tumor antigen. Radiation is thought to trigger the release of potentially immunostimulatory tumor antigens. However, a variety of tumor cells, including human glioblastoma multiforme (GBM) and mouse GL261 glioma cells, express high levels of insulin-like growth factor-1 receptor (IGF-1R), which has been associated with radioresistance. To determine if the combination of targeting IGF-1R with an antisense oligodeoxynucleotide (AS) and radiation causes the release of glioma antigens, we encapsulated GL261 cells in biodiffusion chambers with different doses of IGF-1R AS, subjected the chambers to 5Gy radiation, and cultured the chambers for approximately 18 hours. Bone marrow-derived dendritic cells (BMDCs) from naive mice were pulsed with the cell-free chamber contents and cultured with CD4 T cells from mice protected against the growth of GL261 cells implanted in the cerebral cortex by immunization with a GL261 cell-based vaccine. The numbers of T cells producing IFNγ in an ELISPOT assay were used as an index of antigen recognition. The contents of chambers that were incubated with viable GL261 or were irradiated but cultured without IGF-1R AS stimulated only weak T-cell responses. Substantial T-cell reactivity was only seen with BMDCs pulsed with the cell-free contents of irradiated chambers containing GL261 cells and IGF-1R AS. Overnight treatment of GL261 cells with IGF-1R AS prior to encapsulation and irradiation also promoted antigen release but was most effective only when a small amount of IGF-1R AS was also added to the chambers. Fully formulated chambers contained tumor tissues that had been cultured overnight with the IGF1-R antisense, encapsulated with additional antisense and irradiated. Chambers were incubated in PBS or implanted in the flanks of naive C57BL/6 mice overnight, then contents were retrieved and used to pulse naive BMDCs. CD4 T cells recovered from GL261-immune C57BL/6 mice were used to detect tumor antigenic determinants presented by the BMDCs with the readout being the number of cells producing IFNγ. We conclude that the treatment of IGF-1R-positive glioma cells with IGF-1R AS and sublethal irradiation stimulates the cells to release antigens capable of stimulating the IFNγ-producing CD4 Th1 cells that have been associated with therapeutic immunity against glioma cells in the GL261 model. When performed in a biodiffusion chamber, this process leads to the accumulation of immunostimulatory antigens in the absence of tumor cell products known to have inhibitory properties for Th1 cells. Immunization via the implantation of irradiated biodiffusion chambers containing autologous tumor cells recovered at surgery and IGF-1R AS is currently under investigation in a clinical trial for patients with GBM (IND 14379; ClinicalTrials.gov, NCT01550523). Citation Format: Samantha Garcia, Rhonda Kean, Aurore Lebrun, Emily Bongiorno, David W. Andrews, D. Craig Hooper. Treatment of glioma cells with IGF-1R antisense and irradiation induces the production of antigens that stimulate IFNγ production by tumor-specific CD4 T cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B86.

The liaison librarian to a college with a substantial and growing online learning population began using asynchronous, personalised video instruction as an online replacement for the traditional face-to-face, one-on-one bibliographic instruction reference appointment. This project was informed by the framework of metaliteracy and the “See One, Do One, Teach One” instruction methodology utilised by the health sciences. While formal outcomes assessment has yet to be conducted, unsolicited comments from students are overwhelmingly positive, and preliminary data analysis of usage and engagement reveals several promising trends. 65% of all watched videos were watched for the entire duration of the video, and the liaison librarian found video creation to be less time-consuming than scheduling appointments. The liaison librarian to a university with a substantial and growing online learning population began using asynchronous, personalised video instruction as an online replacement for the traditional face-to-face, one-on-one bibliographic instruction reference appointment. This project was informed by the framework of metaliteracy and the ‘See One, Do One, Teach One’ instruction methodology utilised by the health sciences. While formal outcomes assessment has yet to be conducted, unsolicited comments from students are overwhelmingly positive, and preliminary data analysis of usage and engagement reveals several promising trends. Of all watched videos 65% were watched for the entire duration, and the liaison librarian found video creation to be less time-consuming than scheduling appointments. Providing personalised video instruction tailored to the individual student’s information literacy need is a novel approach that may benefit online learners and librarians alike.

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.

Therapeutic approaches aimed at curing prostate cancer are only partially successful given the occurrence of highly metastatic resistant phenotypes that frequently develop in response to therapies. Recently, we have described α(v)β(6), a surface receptor of the integrin family as a novel therapeutic target for prostate cancer; this epithelial-specific molecule is an ideal target since, unlike other integrins, it is found in different types of cancer but not in normal tissues. We describe a novel α(v)β(6)-mediated signaling pathway that has profound effects on the microenvironment. We show that α(v)β(6) is transferred from cancer cells to monocytes, including β(6)-/- monocytes, by exosomes and that monocytes from prostate cancer patients, but not from healthy volunteers, express α(v)β(6). Cancer cell exosomes, purified via density gradients, promote M2 polarization, whereas α(v)β(6) down-regulation in exosomes inhibits M2 polarization in recipient monocytes. Also, as evaluated by our proteomic analysis, α(v)β(6) down-regulation causes a significant increase in donor cancer cells, and their exosomes, of two molecules that have a tumor suppressive role, STAT1 and MX1/2. Finally, using the Pten(pc-/-) prostate cancer mouse model, which carries a prostate epithelial-specific Pten deletion, we demonstrate that α(v)β(6) inhibition in vivo causes up-regulation of STAT1 in cancer cells. Our results provide evidence of a novel mechanism that regulates M2 polarization and prostate cancer progression through transfer of α(v)β(6) from cancer cells to monocytes through exosomes.

Objectives The United States is experiencing an opioid overdose crisis. Research suggests prolonged postoperative opioid use, a common complication following surgery, is associated with opioid misuse, which, in turn, is the greatest risk factor of heroin misuse. The objective of this review is to evaluate how postoperative opioid exposure relates to prolonged use and to identify factors that predict prolonged postoperative opioid use. Design An integrative review of the literature. Data Sources Electronic and hand searching methods were used in PubMed, Embase, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, CINAHL, and SCOPUS. Search terms included opioid, opiate, postoperative pain, drug administration, prescribing pattern, prescription, inappropriate prescribing, self-medication, patient-controlled analgesia, opioid-naive patients, and prolonged opioid use. Review/Analysis Methods Data were synthesized by identifying themes reflecting the results of the review. A quality assessment of the articles was also conducted. Results Fourteen articles were included and two main themes emerged: (1) Surgery places opioid naive patients at risk for prolonged opioid use and (2) Certain patient characteristics may be predictive of prolonged postoperative opioid use. Conclusions Prolonged postoperative opioid use is related to factors in addition to prescribing practices. Researchers consistently found that patients who are already on opioids, benzodiazepines, or addicted to alcohol; who have mental health disorders, depressive symptoms, or a self-perceived risk of addiction; and patients with multiple co-morbidities are at greater risk of prolonged use; demographics were inconsistent. Nursing Implications Studies are needed to determine the predicting characteristics of prolonged postoperative opioid use, the type of surgeries that place patients at most risk, and the effect postoperative exposure to opioids has on prolonged use. This information can be used to develop and implement protocols to prevent misuse among high-risk patients.

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.

Rabies remains a major public health issue worldwide, especially in developing countries where access to medical care can represent a real challenge. While there is still no cure for rabies, it is a vaccine-preventable disease with pre- and post-exposure prophylaxis regimens approved by the World Health Organization (WHO). However, many rabies-exposed individuals have limited access to vaccines and virus-neutralizing antibodies approved for post-exposure prophylaxis. Unfortunately, any delay in the administration of these reagents can have lethal consequences. This highlights the need to develop cost-effective immunological reagents with a greater window of efficacy. Live-attenuated vaccine strains of rabies virus presents a potential treatment in filling this gap. We show here that immunization with live-attenuated vaccines provide long-lasting rabies immunity, superior to the protection induced by inactivated vaccines. In the absence of an immunostimulatory adjuvant, vaccination with multiple doses of inactivated rabies virus induces a type-2 immune response. This type of immunity is highly effective at inducing neutralizing antibody but has limited efficacy in clearing the virus from central nervous system (CNS) tissues. In contrast, a single infection with live-attenuated rabies vaccine safely drives a type-1 immune response, associated with both the production of a neutralizing antibody and the clearance of wild-type rabies virus from CNS tissues. These results indicate that live-attenuated rabies strains have the potential to be more effective in post-exposure prophylaxis than conventional inactivated vaccines.

Background: We evaluated an autologous cell vaccine, a combination of GBM tumor cells and an antisense molecule against insulin-like growth factor type 1 receptor DNA/mRNA (IGF-1R AS ODN), in adults with newly diagnosed GBM (NCT02507583). Methods: Tumor cells collected during resection were treated ex vivo with IGF-1R AS ODN, encapsulated in biodiffusion chambers with IGF-1R AS ODN, irradiated, then implanted in an abdominal acceptor site on the first post-operative day. Four vaccine exposures were evaluated: lowest (10 chambers implanted for 24 hours); lower (10 / 48 hours); higher (20 / 24 hours); and highest (20 / 48 hours). Standard of care (SOC; ie, radiotherapy and temozolomide) was initiated after 4-6 weeks. Randomization was halted after patient 23 and subsequent patients received the highest exposure. Evaluation of safety and tumor responses were the primary and secondary objectives, respectively. Exploratory objectives included assessment of progression-free survival (PFS) and overall survival (OS). The SOC comparator group was an antecedent cohort of 35 newly diagnosed, GBM patients treated at the same center. Results: Thirty-three patients were enrolled between September 1, 2015 and March 1, 2018. Six, 5, 5, and 17 patients received the lowest, lower, higher, and highest exposures. Median (range) follow-up was 13 (4-39) months. As of the January 1, 2019 cutoff, no vaccine-related adverse events were observed. Seventeen of 33 (51.5%) remained progression-free, 12 of whom are alive and functioning well. The autologous cell vaccine significantly prolonged PFS and OS vs. SOC (Table). Survival advantages were conferred by the highest exposure to the autologous cell vaccine and good T cell function prior to surgery. Conclusions: This vaccine was well-tolerated and prolonged PFS and OS when compared with SOC alone. Table.Survival outcomes in patients receiving vaccine vs. SOC aloneTreatment group2 yr OS estimateMedian OS (mo)p-value v. SOC for OS1 yr PFS estimateMedian PFS estimatep-value v. SOC for PFSVaccine highest dose (N=17)34%21.9.04141%10.4.031Vaccine all (ITT, N=33)31%17.3.01642%9.8.018SOC (n=35)14%12.128%6.9 Citation Format: David W. Andrews, Samantha Garcia, Kevin D. Judy, Larry A. Harshyne, Sanjana Govindarajan, Lawrence Kenyon, Kiran Talekar, Adam Flanders, Kofi-Buaku Atsina, Lyndon Kim, Nina L. Martinez, Wenyin Shi, Maria Werner-Wasik, Mikhail Prosniak, Mark T. Curtis, Rhonda Kean, Emily Bongiorno, Sami Sauma, Kara Pigott, Charles B. Scott, D Craig Hooper. Results of a Phase Ib trial of an autologous cell vaccine for newly diagnosed glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT038.

Pathological changes occur in areas of CNS tissue remote from inflammatory lesions in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). To determine if oxidative stress is a significant contributor to this non-inflammatory pathology, cortex tissues from mice with clinical signs of EAE were examined for evidence of inflammation and oxidative stress. Histology and gene expression analysis showed little evidence of immune/inflammatory cell invasion but reductions in natural antioxidant levels and increased protein oxidation that paralleled disease severity. Two-dimensional oxyblots and mass-spectrometry-based protein fingerprinting identified glutamine synthetase (GS) as a particular target of oxidation. Oxidation of GS was associated with reductions in enzyme activity and increased glutamate/glutamine levels. The possibility that this may cause neurodegeneration through glutamate excitotoxicity is supported by evidence of increasing cortical Ca(2+) levels in cortex extracts from animals with greater disease severity. These findings indicate that oxidative stress occurs in brain areas that are not actively undergoing inflammation in EAE and that this can lead to a neurodegenerative process due to the susceptibility of GS to oxidative inactivation.

The loss of blood-brain barrier (BBB) integrity in CNS inflammatory responses triggered by infection and autoimmunity has generally been associated with the development of neurological signs. In the present study, we demonstrate that the clearance of the attenuated rabies virus CVS-F3 from the CNS is an exception; increased BBB permeability and CNS inflammation occurs in the absence of neurological sequelae. We speculate that regionalization of the CNS inflammatory response contributes to its lack of pathogenicity. Despite virus replication and the expression of several chemokines and IL-6 in both regions being similar, the up-regulation of MIP-1β, TNF-α, IFN-γ, and ICAM-1 and the loss of BBB integrity was more extensive in the cerebellum than in the cerebral cortex. The accumulation of CD4- and CD19-positive cells was higher in the cerebellum than the cerebral cortex. Elevated CD19 levels were paralleled by κ-L chain expression levels. The timing of BBB permeability changes, κ-L chain expression in CNS tissues, and Ab production in the periphery suggest that the in situ production of virus-neutralizing Ab may be more important in virus clearance than the infiltration of circulating Ab. The data indicate that, with the possible exception of CD8 T cells, the effectors of rabies virus clearance are more commonly targeted to the cerebellum. This is likely the result of differences in the capacity of the tissues of the cerebellum and cerebral cortex to mediate the events required for BBB permeability changes and cell invasion during virus infection.

Urate (UA) selectively scavenges peroxynitrite-dependent radicals and suppresses CNS inflammation through effects that are manifested at the blood-brain barrier (BBB). ICAM-1 upregulation in the spinal cord tissues of myelin basic protein (MBP) immunized mice is selectively inhibited by UA treatment. In contrast, the expression of ICAM-1 and other adhesion molecules by circulating cells is unchanged. Moreover, TNF-alpha expression in the CNS tissues of MBP-immunized mice is suppressed by UA treatment but TNF-alpha-induced ICAM-1 expression on neurovascular endothelial cells is not. Therefore the effect of UA on ICAM-1 upregulation in the CNS tissues is likely due to its known contribution to the maintenance of BBB integrity in MBP-immunized mice which in turn inhibits cell invasion into the CNS and prevents TNF-alpha production in CNS tissues.

e16058 Background: Preclinical data suggest there are synergistic effects of radiation therapy (RT) and check point inhibitors in anticancer immunity. The primary objective of this study was to explore the immunomodulatory activity of RT alone or in combination with Pembrolizumab (pembro) in solid tumors including renal cell cancer (RCC) patients (pts). Methods: RCC pts who progressed after at least one front-line therapy were eligible. Pts were treated with either RT (8Gy x 1 or 4Gy x 5) followed by (f/b)pembro or 1 dose pembro f/b RT f/b pembro. Pre- and post RT tumor biopsy was obtained to evaluate PD-L1 expression (assay by QualTeck). Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry. Treatment response was measured based on modified RECIST criteria. Results: Twelve RCC patients were enrolled including 2 with non-clear cell subtype. One pt was not evaluable since pt quickly deteriorated and was taken off study. As of January 13, 2017, 2 pts are still on active treatment. Two pts had partial response (18%) and were on study for at 54 and 63 weeks. One responder was treated with 8 Gy f/b pembro while 1 was treated with 1 dose pembro f/b RT f/b pembro. Five pts had stable disease of 18 to 45 weeks and 4 pts (36%, non-responders) had progression in 9 weeks. For adverse events, 1 pt developed grade 3 pneumonitis after 10 cycles of pembro (RT to adrenal mets). Grade 3 AEs include Fatigue, Nausea, Hyperglycemia, Lymphopenia, thrombocytopenia and AST elevation (post RT for liver mets). PDL1 expression and tumor infiltrating lymphocytes presence after RT showed various patterns. Preliminary flow cytometry showed persistent higher numbers of monocytes in non-responders comparing with responders. CD4+, CD8+ and NK cells and other markers are under analyzed and the results will be presented. Conclusions: The combination of RT (8Gy or 20Gy) with pembro is feasible and tolerated, and demonstrates clinical activity. The AE profile is similar to single agent pembro. Monocytes, T and NK cell kinetics are being examined. (ClinicalTrials.gov ID: NCT02318771) Clinical trial information: NCT02318771.

Raytheon continues to build large format digital visible focal planes. This paper provides the most recent performance to date in operability and performance.

Uric acid (UA), a product of purine metabolism, is a known scavenger of peroxynitrite (ONOO−), which has been implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis (EAE). To determine whether the known therapeutic action of UA in EAE is mediated through its capacity to inactivate ONOO− or some other immunoregulatory phenomenon, the effects of UA on Ag presentation, T cell reactivity, Ab production, and evidence of CNS inflammation were assessed. The inclusion of physiological levels of UA in culture effectively inhibited ONOO−-mediated oxidation as well as tyrosine nitration, which has been associated with damage in EAE and multiple sclerosis, but had no inhibitory effect on the T cell-proliferative response to myelin basic protein (MBP) or on APC function. In addition, UA treatment was found to have no notable effect on the development of the immune response to MBP in vivo, as measured by the production of MBP-specific Ab and the induction of MBP-specific T cells. The appearance of cells expressing mRNA for inducible NO synthase in the circulation of MBP-immunized mice was also unaffected by UA treatment. However, in UA-treated animals, the blood-CNS barrier breakdown normally associated with EAE did not occur, and inducible NO synthase-positive cells most often failed to reach CNS tissue. These findings are consistent with the notion that UA is therapeutic in EAE by inactivating ONOO−, or a related molecule, which is produced by activated monocytes and contributes to both enhanced blood-CNS barrier permeability as well as CNS tissue pathology.

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.

Viral insults that occur during early postnatal periods, can affect neuronal systems which exhibit significant postnatal development, such as the cerebral cortex and cerebellum. Borna disease virus (BDV) is a single-strand RNA virus which replicates in the nervous system of many species after experimental inoculation and causes acute neurological disease. Neonatal rats infected with BDV do not mount an aggressive response to the virus like their adult counterparts, but instead develop a persistent BDV infection with less overt clinical sequelae. Recently, the cerebellum, a neural structure associated with regulation of motor behavior, and perhaps with higher cognitive functions, has been demonstrated to be a target of neonatal BDV infections in rats (Bautista et al, 1995). In the present study neonatal rats were infected with BDV and their cerebella were analyzed histologically and immunohistochemically at 7 months of age. The cerebella of infected animals were reduced in size but normal foliation and laminar organization was present. However, as visualized with immunohistochemistry for the Purkinje cell-specific antigen calbindin, there were numerous gaps within the Purkinje cell layer and in the molecular layer which contains the Purkinje cell dendritic trees. We estimated the number of Purkinje cells and found there was an approximately 75% loss of PC in adult rats neonatally infected with BDV. These results suggest that neonatal BDV infection may either (1) target the PC and cause the death of these cells directly or (2) acts indirectly by triggering an immune response which is then responsible for the loss of these cells.

Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-γ receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.

Because of the short half-life of NO, previous studies implicating NO in central nervous system pathology during infection had to rely on the demonstration of elevated levels of NO synthase mRNA or enzyme expression or NO metabolites such as nitrate and nitrite in the infected brain. To more definitively investigate the potential causative role of NO in lesions of the central nervous system in animals infected with neurotropic viruses or suffering from experimental allergic encephalitis, we have determined directly the levels of NO present in the central nervous system of such animals. Using spin trapping of NO and electron paramagnetic resonance spectroscopy, we confirm here that copious amounts of NO (up to 30-fold more than control) are elaborated in the brains of rats infected with rabies virus or borna disease virus, as well as in the spinal cords of rats that had received myelin basic protein-specific T cells.

A single intramuscular application of the live but not UV-inactivated recombinant rabies virus (RABV) variant TriGAS in mice induces the robust and sustained production of RABV-neutralizing antibodies that correlate with long-term protection against challenge with an otherwise lethal dose of the wild-type RABV. To obtain insight into the mechanism by which live TriGAS induces long-lasting protective immunity, quantitative PCR (qPCR) analysis of muscle tissue, draining lymph nodes, spleen, spinal cord, and brain at different times after TriGAS inoculation revealed the presence of significant copy numbers of RABV-specific RNA in muscle, lymph node, and to a lesser extent, spleen for several days postinfection. Notably, no significant amounts of RABV RNA were detected in brain or spinal cord at any time after TriGAS inoculation. Differential qPCR analysis revealed that the RABV-specific RNA detected in muscle is predominantly genomic RNA, whereas RABV RNA detected in draining lymph nodes is predominantly mRNA. Comparison of genomic RNA and mRNA obtained from isolated lymph node cells showed the highest mRNA-to-genomic-RNA ratios in B cells and dendritic cells (DCs), suggesting that these cells represent the major cell population that is infected in the lymph node. Since RABV RNA declined to undetectable levels by 14 days postinoculation of TriGAS, we speculate that a transient infection of DCs with TriGAS may be highly immunostimulatory through mechanisms that enhance antigen presentation. Our results support the superior efficacy and safety of TriGAS and advocate for its utility as a vaccine.

OBJECTIVES The purpose of the study was to assess the impact that funding from the National Network of Libraries of Medicine (NN/LM), Greater Midwest Region (GMR), has on member institutions' ability to conduct outreach on behalf of NN/LM. METHODS The study employed both content analysis and survey methodologies. The final reports from select GMR-funded outreach projects (n = 20) were analyzed based on a set of evaluation criteria. Project principal investigators (n = 13) were then surveyed using the same evaluation criteria. RESULTS Results indicated that outreach projects supported by GMR funding improved access to biomedical information for professionals and the general public. Barriers to conducting outreach projects included time constraints or commitments, staffing, scheduling and absenteeism, inadequate space, and issues associated with technology (e.g., hardware and software, Internet connectivity and firewall issues, and creation and use of new technologies). CONCLUSIONS The majority of project principal investigators indicated that their attempts to conduct outreach were successful. Moreover, most noted that outreach had a positive impact on professionals as well as the general public. In general, it seems that negative outcomes, as with most barriers to conducting outreach, can be mitigated by more thorough planning.

The long-held concept that rabies infection is lethal in humans once the causative rabies virus has reached the CNS has been called into question by the recent survival of a number of patients with clinical rabies. Studies in animal models provide insight into why survival from a rabies virus infection that has spread to the CNS is possible and the immune mechanisms involved. In the CNS, both innate mechanisms capable of inhibiting virus replication and the activity of infiltrating rabies virus-specific T and B cells with the capacity to clear the virus are required. Deficiencies in the induction of either aspect of rabies immunity can lead to lethal consequences but may be overcome by novel approaches to active and passive immunization.

Rabies, a neurological disease associated with replication in central nervous system (CNS) tissues of any of a number of rabies viruses endemic in nature, is generally fatal. Prophylactic medical intervention is immune mediated and directed at preventing the spread of the virus from a peripheral site of exposure to the CNS. While individuals rarely develop immune responses capable of clearing the virus from CNS tissues, a variety of laboratory-attenuated rabies viruses are readily cleared from the CNS tissues in animal models. By comparing immune responses to wild-type and attenuated rabies viruses in these models, we have discovered that the latter induce processes required for immune effector infiltration into CNS tissues that are absent from lethal infections. Predominant among these are activities of cells of the neurovascular unit (NVU) that promote an interaction with circulating immune cells. In the absence of this interaction, the specialized barrier function of the NVU remains intact and circulating virus-specific immune effectors are largely excluded from infected CNS tissues. Studies of mixed infections with wild-type and attenuated rabies viruses reveal that wild-type rabies viruses fail to trigger, rather than inhibit, the interactions between immune cells and the NVU required for virus clearance from the CNS. These studies provide insights into how immune effectors with the capacity to clear the virus may be delivered into CNS tissues to contain a wild-type rabies virus infection. However, to apply immunotherapeutic strategies beyond the initial stages of CNS infection, further insights into the fate of the infected cells during virus clearance are needed.

Investigations were conducted to determine the nature and cause of magnetic field disturbances at the calibration hardstand (compass rose) at an air force base. The disturbances have prevented the normal and routine use of the hardstand since its construction in the mid-1950s, with heightened concern over the problem every 10–12 years. The investigations included the characterization of the horizontal, vertical, and temporal variation of the total magnetic field strength both on and off the hardstand. On the hardstand, the field varies by as much as 1,000 nT over 10 m horizontally and by as much as 600 nT over 2.5 m vertically. Also, on the hardstand, the magnetic field varies extremely erratically with time by 50–60 nT over periods of a few seconds. Off the hardstand, the magnetic field is extremely stable, and varies by less than 10 nT over 10 m horizontally, 2.5 m vertically, and over periods of 10–15 min. The magnetic field “stabilizes” at distances less than 10 m horizontally from the edge of the hardstand. Both on and off the hardstand, the magnetic field variations are independent of the status (on or off) of a nearby instrument landing system (ILS) (approach radar). The results of the magnetic field characterization indicate that the cause of the problem is the hardstand itself. The hardstand concrete was found to have a large bulk magnetic susceptibility, and the concrete aggregate has a permanent magnetization sufficiently large to visibly deflect a compass needle; these factors, coupled with a correlation with the 11-year solar cycle, suffice to explain the magnetic “disturbances” at the hardstand.

Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.

Vignettes depicting the physical mistreatment, psychological mistreatment and neglect of children were used in an experimental design to test for bias in perception and reportage of child abuse. Subjects were 160 jurors and 176 child protective service (CPS) and police personnel. Jurors born before 1945 were less critical of such mistreatment than those born later. Female jurors pledged to report mistreatment more often than did male jurors, and they pledged to report the abuse of a female victim more often than a male victim. The other groups did not show this bias in reporting. Findings might explain discrepancies between studies of the incidence of child abuse and reportage of it.

CNS tissues are protected from circulating cells and factors by the blood–brain barrier (BBB), a specialization of the neurovasculature. Outcomes of the loss of BBB integrity and cell infiltration into CNS tissues can differ vastly. For example, elevated BBB permeability is closely associated with the development of neurological disease in experimental allergic encephalomyelitis (EAE) but not during clearance of the attenuated rabies virus CVS-F3 from the CNS tissues. To probe whether differences in the nature of BBB permeability changes may contribute to the pathogenesis of acute neuroinflammatory disease, we compared the characteristics of BBB permeability changes in mice with EAE and in mice clearing CVS-F3. BBB permeability changes are largely restricted to the cerebellum and spinal cord in both models but differ in the extent of leakage of markers of different size and in the nature of cell accumulation in the CNS tissues. The accumulation in the CNS tissues of CD4 T cells expressing mRNAs specific for IFN-γ and IL-17 is common to both, but iNOS-positive cells invade into the CNS parenchyma only in EAE. Mice that have been immunized with myelin basic protein (MBP) and infected exhibit the features of EAE. Treatment with the peroxynitrite-dependent radical scavenger urate inhibits the invasion of iNOS-positive cells into the CNS tissues and the development of clinical signs of EAE without preventing the loss of BBB integrity in immunized/infected animals. These findings indicate that BBB permeability changes can occur in the absence of neuropathology provided that cell invasion is restricted.

The blood-brain barrier (BBB) is dramatically but transiently compromised in the cerebella of myelin basic protein immunized mice at least one week prior to the development of the paralytic phase of experimental allergic encephalomyelitis (EAE). Treatment of mice with the peroxynitrite-dependent radical scavenger uric acid (UA) during the first week after immunization blocks the early increase in cerebellar BBB permeability and the subsequent development of clinical signs of EAE. These results indicate that the early loss of BBB integrity in the cerebellum is likely to be a necessary step in the development of paralytic EAE.

Elevated blood-brain barrier (BBB) permeability is associated with both the protective and pathological invasion of immune and inflammatory cells into CNS tissues. Although a variety of processes have been implicated in the changes at the BBB that result in the loss of integrity, there has been no consensus as to their induction. TNF-α has often been proposed to be responsible for increased BBB permeability but there is accumulating evidence that peroxynitrite (ONOO−)-dependent radicals may be the direct trigger. We demonstrate here that enhanced BBB permeability in mice, whether associated with rabies virus (RV) clearance or CNS autoimmunity, is unaltered in the absence of TNF-α. Moreover, the induction of TNF-α expression in CNS tissues by RV infection has no impact on BBB integrity in the absence of T cells. CD4 T cells are required to enhance BBB permeability in response to the CNS infection whereas CD8 T cells and B cells are not. Like CNS autoimmunity, elevated BBB permeability in response to RV infection is evidently mediated by ONOO−. However, as opposed to the invading cells producing ONOO− that have been implicated in the pathogenesis of CNS inflammation, during virus clearance ONOO− is produced without pathological sequelae by IFN-γ-stimulated neurovascular endothelial cells.

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is used to model certain parameters of multiple sclerosis. To establish the relative contributions of T cell reactivity, the loss of blood-brain barrier (BBB) integrity, CNS inflammation, and lesion formation toward the pathogenesis of EAE, we assessed the incidence of EAE and these parameters in mice lacking NF-kappaB, TNF-alpha, IFN-alphabeta receptors, IFN-gamma receptors, and inducible nitric oxide synthase. Although increased myelin oligodendrocyte glycoprotein-specific T cell reactivity was generally associated with a more rapid onset or increased disease severity, the loss of BBB integrity and cell accumulation in spinal cord tissues was invariably associated with the development of neurological disease signs. Histological and real-time RT-PCR analyses revealed differences in the nature of immune/inflammatory cell accumulation in the spinal cord tissues of the different mouse strains. On the other hand, disease severity during the acute phase of EAE directly correlated with the extent of BBB permeability. Thus, the loss of BBB integrity seems to be a requisite event in the development of EAE and can occur in the absence of important inflammatory mediators.

Poly(ADP-ribose) polymerase (PARP) activity has been implicated in the pathogenesis of several central nervous system (CNS) disorders. For example, the presence of extensive poly(ADP)ribosylation in CNS tissues from animals with experimental allergic encephalomyelitis (EAE) indicates that PARP activity may be involved in this inflammatory disease process. Using PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl], a selective PARP inhibitor, we studied the mechanisms through which PARP activity may contribute to the onset of acute EAE. PLSJL mice immunized with myelin antigens were treated with PJ34, and the effects on the progression of EAE and several other parameters relevant to the disease process were assessed. PJ34 exerted therapeutic effects at the onset of EAE that were associated with reduced CNS inflammation and the maintenance of neurovascular integrity. Expression of genes encoding the intercellular adhesion molecule-1 (ICAM-1) and the inflammatory mediators interferon-gamma, tumor necrosis factor-alpha, and inducible nitric-oxide synthase were decreased in CNS tissues from drug-treated animals. Administration of PJ34 biased the class of myelin basic protein (MBP)-specific antibodies elicited from IgG2a to IgG1 and IgG2b and modulated antigen-specific T-cell reactivity. Therefore, the mode of action of PJ34 at the onset of EAE is likely mediated by a shift in the MBP-specific immune response from a proinflammatory Th1 toward an anti-inflammatory Th2 phenotype.

Serum levels of uric acid (UA), an inhibitor of peroxynitrite- (ONOO − ) related chemical reactions, became elevated approximately 30 million years ago in hominid evolution. During a similar time frame, higher mammals lost the ability to synthesize another important radical scavenger, ascorbic acid (AA), leading to the suggestion that UA may have replaced AA as an antioxidant. However, in vivo treatment with AA does not protect against the development of experimental allergic encephalomyelitis (EAE), a disease that has been associated with the activity of ONOO − and is inhibited by UA. When compared in vitro, UA and AA were found to have similar capacities to inhibit the nitrating properties of ONOO − . However UA and AA had different capacities to prevent ONOO − -mediated oxidation, especially in the presence of iron ion (Fe 3+ ). While UA at physiological concentrations effectively blocked dihydrorhodamine-123 oxidation in the presence of Fe 3+ , AA did not, regardless of whether the source of ONOO − was synthetic ONOO − , SIN-1, or RAW 264.7 cells. AA also potentiated lipid peroxidation in vivo and in vitro. In conclusion, the superior protective properties of UA in EAE may be related to its ability to neutralize the oxidative properties of ONOO − in the presence of free iron ions.

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253–275) and nucleoprotein (N protein) (amino acids 404–418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.

Peroxynitrite has been implicated in the pathogenesis of multiple sclerosis and its animal counterpart experimental allergic encephalomyelitis (EAE). Here we have examined the effects of the novel peroxynitrite scavengers, mercaptoethylguanidine (MEG) and guanidinoethyldisulphide (GED), on the development of EAE. Both MEG and GED delayed EAE onset and decreased the number of animals displaying disease signs. However, when EAE developed, its severity was not significantly abrogated by drug administration. These results suggest that while MEG and GED protect against the induction phase of EAE, they do not prevent disease progression. This may be due to the inability of MEG and GED to efficiently scavenge peroxynitrite or result from their capacity to inhibit inducible nitric oxide synthase. Therefore, the development of more potent and selective scavengers of peroxynitrite is necessary for use in EAE.