Validation of the clinical utility of sGaw as a response variable in methacholine challenge testing.

BACKGROUND AND OBJECTIVE: Airway hyperresponsiveness (AHR) is commonly assessed by a methacholine challenge test (MCT), during which a provocative concentration causing a 20% reduction in forced expiratory volume in 1 second (FEV1 ) (PC20 ) < 8 mg/ml is considered a positive response. However, a fall in specific airway conductance (sGaw) may also have clinical significance. The purpose of this study was to assess whether AHR determined by a provocative concentration causing a 40% reduction in sGaw (PC40 ) < 8 mg/ml corresponds to a clinical diagnosis of asthma. METHODS: We analysed the changes in spirometry, lung volumes and sGaw during MCT in 211 randomly selected patients being evaluated for AHR to support a clinical diagnosis of asthma. RESULTS: The mean (SD) age of the group was 53 (15) years, with 141 women (67%). Overall lung function was normal, with FEV1  = 92 (15) % predicted, total lung capacity = 97 (13) % predicted and sGaw = 0.19 (0.15-0.23) L/s/cm H2 O/L, (median, 25-75 IQR). There were many more patients who responded by PC40 only (n = 120) than who responded by PC20 (n = 52). There was no significant difference in asthma diagnosis between the PC20 (98%) and PC40 (93%) groups, and we estimate 34% of patients with a diagnosis of asthma would have been classified as having no AHR if only the FEV1 criterion was used. CONCLUSION: Changes in sGaw during MCT indicate clinically significant AHR in support of a clinical diagnosis of asthma among patients being evaluated for asthma.

Hepcidin Is Essential for Alveolar Macrophage Function and Is Disrupted by Smoke in a Murine Chronic Obstructive Pulmonary Disease Model.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

The Role of Dopamine in the Collective Regulation of Foraging in Harvester Ants.

Colonies of the red harvester ant (Pogonomyrmex barbatus) differ in how they regulate collective foraging activity in response to changes in humidity. We used transcriptomic, physiological, and pharmacological experiments to investigate the molecular basis of this ecologically important variation in collective behavior among colonies. RNA sequencing of forager brain tissue showed an association between colony foraging activity and differential expression of transcripts related to biogenic amine and neurohormonal metabolism and signaling. In field experiments, pharmacological increases in forager brain dopamine titer caused significant increases in foraging activity. Colonies that were naturally most sensitive to humidity were significantly more responsive to the stimulatory effect of exogenous dopamine. In addition, forager brain tissue significantly varied among colonies in biogenic amine content. Neurophysiological variation among colonies associated with individual forager sensitivity to humidity may reflect the heritable molecular variation on which natural selection acts to shape the collective regulation of foraging.

Efficacy of Low-Dose Buspirone for Restricted and Repetitive Behavior in Young Children with Autism Spectrum Disorder: A Randomized Trial.

OBJECTIVES: To determine safety and efficacy of the 5HT1A serotonin partial agonist buspirone on core autism and associated features in children with autism spectrum disorder (ASD). STUDY DESIGN: Children 2-6 years of age with ASD (N = 166) were randomized to receive placebo or 2.5 or 5.0 mg of buspirone twice daily. The primary objective was to evaluate the effects of 24 weeks of buspirone on the Autism Diagnostic Observation Schedule (ADOS) Composite Total Score. Secondary objectives included evaluating the effects of buspirone on social competence, repetitive behaviors, language, sensory dysfunction, and anxiety and to assess side effects. Positron emission tomography measures of tryptophan metabolism and blood serotonin concentrations were assessed as predictors of buspirone efficacy. RESULTS: There was no difference in the ADOS Composite Total Score between baseline and 24 weeks among the 3 treatment groups (P = .400); however, the ADOS Restricted and Repetitive Behavior score showed a time-by-treatment effect (P = .006); the 2.5-mg buspirone group showed significant improvement (P = .003), whereas placebo and 5.0-mg buspirone groups showed no change. Children in the 2.5-mg buspirone group were more likely to improve if they had fewer foci of increased brain tryptophan metabolism on positron emission tomography (P = .018) or if they showed normal levels of blood serotonin (P = .044). Adverse events did not differ significantly among treatment groups. CONCLUSIONS: Treatment with 2.5 mg of buspirone in young children with ASD might be a useful adjunct therapy to target restrictive and repetitive behaviors in conjunction with behavioral interventions. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00873509.

A phase 1 trial of oncolytic HSV-1, G207, given in combination with radiation for recurrent GBM demonstrates safety and radiographic responses.

G207, a mutant herpes simplex virus (HSV) type 1, is safe when inoculated into recurrent malignant glioma. We conducted a phase 1 trial of G207 to demonstrate the safety of stereotactic intratumoral administration when given 24 hours prior to a single 5 Gy radiation dose in patients with recurrent malignant glioma. Nine patients with progressive, recurrent malignant glioma despite standard therapy were included. Patients received one dose of G207 stereotactically inoculated into the multiple sites of the enhancing tumor margin and were then treated focally with 5 Gy radiation. Treatment was well tolerated, and no patient developed HSV encephalitis. The median interval between initial diagnosis and G207 inoculation was 18 months (mean: 23 months; range: 11-51 months). Six of the nine patients had stable disease or partial response for at least one time point. Three instances of marked radiographic response to treatment occurred. The median survival time from G207 inoculation until death was 7.5 months (95% confidence interval: 3.0-12.7). In conclusion, this study showed the safety and the potential for clinical response of single-dose oncolytic HSV therapy augmented with radiation in the treatment of malignant glioma patients. Additional studies with oncolytic HSV such as G207 in the treatment of human glioma are recommended.

Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a conditionally-replicating oncolytic HSV-1.

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.

CD133 marks a myogenically primitive subpopulation in rhabdomyosarcoma cell lines that are relatively chemoresistant but sensitive to mutant HSV.

BACKGROUND: Rhabdomyosarcoma (RMS) is characterized by features of skeletal muscle and is comprised of two major histological subtypes, embryonal (E-RMS), and alveolar (A-RMS). Subsets of each RMS subtype demonstrate resistance to multimodal therapy leading to treatment failure. Cancer stem cells or cancer-initiating cells (CIC) represent a theorized population of cells that give rise to tumors and are responsible for treatment resistance. PROCEDURE: We investigated the ability of CD133, a putative CIC marker, to distinguish a chemoresistant, myogenically primitive population in alveolar (RH30), and embryonal (RD) RMS cell lines. We tested CD133+/- cells for sensitivity to engineered herpes simplex virus (oHSV). RESULTS: Relative to CD133- cells, CD133+ A-RMS, and E-RMS cells demonstrate an enhanced colony-forming ability, are less differentiated myogenically, and are more resistant to cytotoxic chemotherapy but equally sensitive to oHSV oncolysis. Compared to CD133- RD cells, CD133+ cells express relatively high levels of genes typically expressed in skeletal muscle progenitor satellite cells including PAX7, c-MET, and the GLI effectors of the hedgehog signaling pathway. In contrast, CD133+ RH30 cells were not associated with enhanced expression of satellite cell markers or Hh targets. CONCLUSIONS: Our findings demonstrate that CD133+ cells from A-RMS and E-RMS cell lines are characterized by a myogenically primitive phenotype. These cells have the capacity to form colonies in vitro and are more resistant to chemotherapy than CD133- cells. CD133 expression may denote a subset of RMS cells with an important role in tumorigenesis and treatment failure. These resistant cells may be effectively targeted by oHSV therapy.

Preclinical evaluation of a genetically engineered herpes simplex virus expressing interleukin-12.

Herpes simplex virus 1 (HSV-1) mutants that lack the γ(1)34.5 gene are unable to replicate in the central nervous system but maintain replication competence in dividing cell populations, such as those found in brain tumors. We have previously demonstrated that a γ(1)34.5-deleted HSV-1 expressing murine interleukin-12 (IL-12; M002) prolonged survival of immunocompetent mice in intracranial models of brain tumors. We hypothesized that M002 would be suitable for use in clinical trials for patients with malignant glioma. To test this hypothesis, we (i) compared the efficacy of M002 to three other HSV-1 mutants, R3659, R8306, and G207, in murine models of brain tumors, (ii) examined the safety and biodistribution of M002 in the HSV-1-sensitive primate Aotus nancymae following intracerebral inoculation, and (iii) determined whether murine IL-12 produced by M002 was capable of activating primate lymphocytes. Results are summarized as follows: (i) M002 demonstrated superior antitumor activity in two different murine brain tumor models compared to three other genetically engineered HSV-1 mutants; (ii) no significant clinical or magnetic resonance imaging evidence of toxicity was observed following direct inoculation of M002 into the right frontal lobes of A. nancymae; (iii) there was no histopathologic evidence of disease in A. nancymae 1 month or 5.5 years following direct inoculation; and (iv) murine IL-12 produced by M002 activates A. nancymae lymphocytes in vitro. We conclude that the safety and preclinical efficacy of M002 warrants the advancement of a Δγ(1)34.5 virus expressing IL-12 to phase I clinical trials for patients with recurrent malignant glioma.

Preclinical evaluation of oncolytic δγ(1)34.5 herpes simplex virus expressing interleukin-12 for therapy of breast cancer brain metastases.

The metastasis of breast cancer to the brain and central nervous system (CNS) is a problem of increasing importance. As improving treatments continue to extend patient survival, the incidence of CNS metastases from breast cancer is on the rise. New treatments are needed, as current treatments are limited by deleterious side effects and are generally palliative. We have previously described an oncolytic herpes simplex virus (HSV), designated M002, which lacks both copies of the γ(1)34.5 neurovirulence gene and carries a murine interleukin 12 (IL-12) expression cassette, and have validated its antitumor efficacy in a variety of preclinical models of primary brain tumors. However, M002 has not been yet evaluated for use against metastatic brain tumors. Here, we demonstrate the following: both human breast cancer and murine mammary carcinoma cells support viral replication and IL-12 expression from M002; M002 replicates in and destroys breast cancer cells from a variety of histological subtypes, including "triple-negative" and HER2 overexpressing; M002 improves survival in an immunocompetent model more effectively than does a non-cytokine control virus. Thus, we conclude from this proof-of-principle study that a γ(1)34.5-deleted IL-12 expressing oncolytic HSV may be a potential new therapy for breast cancer brain metastases.

Posttranscriptional control of type I interferon genes by KSRP in the innate immune response against viral infection.

Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3' untranslated regions. Expression of ARE-containing type I interferon transcripts is robustly induced upon viral infection and rapidly shut off thereafter. Their transient accumulation is partly mediated through posttranscriptional regulation. Here we show that mouse embryonic fibroblasts derived from knockout mice deficient in KH-type splicing regulatory protein (KSRP), an RNA-binding protein required for ARE-mediated mRNA decay, produce higher levels of Ifna and Ifnb mRNAs in response to viral infection as a result of decreased mRNA decay. Functional analysis showed that KSRP is required for the decay of Ifna4 and Ifnb mRNAs by interaction with AREs. The increased IFN expression renders Ksrp(-)(/)(-) cells refractory to herpes simplex virus type 1 and vesicular stomatitis virus infection. These findings support a role of a posttranscriptional mechanism in the control of type I IFN expression and highlight the function of KSRP in innate immunity by negatively regulating IFN production.

Strategies for the rapid construction of conditionally-replicating HSV-1 vectors expressing foreign genes as anticancer therapeutic agents.

Conditionally replication-competent Herpes Simplex Virus Type 1 (HSV-1) vectors expressing foreign genes have been developed as experimental therapeutic agents. Traditional methods of virus construction, including growth selection based on thymidine kinase gene expression, and color selection based on a reporter gene expression are often time-consuming and relatively inefficient. This review summarizes the various strategies developed in recent years for the rapid and efficient construction of novel conditionally replication-competent mutant HSV expressing multiple foreign genes. Additionally, two new modifications of existing strategies, which have not been previously reported, are discussed.

Herpesvirus vectors for therapy of brain tumors.

Genetically modified, conditionally-replicating Herpes Simplex Virus Type 1 (HSV-1) vectors for the treatment of malignant glioma have provided encouraging results in the handful of Phase I and Phase II clinical trials conducted to date. In recent years, a number of new strategies have been developed to improve anti-tumor activity of these attenuated vectors, through either introduction of foreign gene inserts to enhance tumor killing through a variety of mechanisms, or through combination with existing treatment regimens, including radiation and/or chemotherapeutics. Another promising new approach has been the engineering of novel oncolytic HSV vectors that retain wildtype replication, but are targeted to tumor cells through a variety of mechanisms. This review summarizes the latest advances in herpesvirus-mediated oncolytic therapies from both preclinical results and clinical trials with oncolytic HSV vectors in patients, and their implication for design of future trials.

Oncolytic viral therapy of malignant glioma.

Novel approaches to treatment of malignant glioma, the most frequently occurring primary brain tumor, have included the use of a wide range of oncolytic viral vectors. These vectors, either naturally tumor-selective, or engineered as such, have shown promise in the handful of phase I and phase II clinical trials conducted in recent years. The strategies developed for each of the different viruses currently being studied and the history of their development are summarized here. In addition, the results of clinical trials in patients and their implication for future trials are also discussed.

Engineered herpes simplex viruses efficiently infect and kill CD133+ human glioma xenograft cells that express CD111.

Oncolytic herpes simplex viruses (HSV) hold promise for therapy of glioblastoma multiforme (GBM) resistant to traditional therapies. We examined the ability of genetically engineered HSV to infect and kill cells that express CD133, a putative marker of glioma progenitor cells (GPC), to determine if GPC have an inherent therapeutic resistance to HSV. Expression of CD133 and CD111 (nectin-1), the major entry molecule for HSV, was variable in six human glioma xenografts, at initial disaggregation and after tissue culture. Importantly, both CD133+ and CD133- populations of glioma cells expressed CD111 in similar relative proportions in five xenografts, and CD133+ and CD133- glioma cell subpopulations were equally sensitive to killing in vitro by graded dilutions of wild-type HSV-1(F) or several different gamma(1)34.5-deleted viruses. GPC did not display an inherent resistance to HSV. While CD111 expression was an important factor for determining sensitivity of glioma cells to HSV oncolysis, it was not the only factor. Our findings support the notion that HSV will not be able to effectively enter, infect, and kill cells in tumors that have low CD111 expression (<20%). However, virotherapy with HSV may be very effective against CD111+ GPC resistant to traditional therapies.

Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-and post-tumor resection for recurrent GBM.

We have previously demonstrated safety of G207, a doubly mutated (deletion of both gamma(1)34.5 loci, insertional inactivation of U(L)39) herpes simplex virus (HSV) for patients stereotactically inoculated in enhancing portions of recurrent malignant gliomas. We have now determined safety of two inoculations of G207, before and after tumor resection. Inclusion criteria were histologically proven recurrent malignant glioma, Karnofsky score >or=70, and ability to resect the tumor without ventricular system breach. Patients received two doses of G207 totaling 1.15 x 10(9) plaque-forming units with 13% of this total injected via a catheter placed stereotactically in the tumor. Two or five days later, tumor was resected en bloc with catheter in place. The balance of G207 dose was injected into brain surrounding the resection cavity. Six patients with recurrent glioblastoma multiforme were enrolled. Two days after the second G207 inoculation, one patient experienced transient fever, delirium, and hemiparesis, which entirely resolved on high-dose dexamethasone. No patient developed HSV encephalitis or required treatment with acyclovir. Radiographic and neuropathologic evidence suggestive of antitumor activity is reported. Evidence of viral replication was demonstrated. G207 appears safe for multiple dose delivery, including direct inoculation into the brain surrounding tumor resection cavity.

Spontaneous and Engineered Compensatory HSV Mutants that Counteract the Host Antiviral PKR Response.

A virulent recombinant HSV lacking the diploid γ(1)34.5 gene (Δγ(1)34.5) have been investigated over the last two decades both for anti-tumor therapy and as vaccine vectors. The first generation vectors, while safe, are incapable of sustained replication in the majority of treated patients. An interferon inducible host antiviral kinase, protein kinase R (PKR), limits late viral protein synthesis and replication of Δγ(1)34.5 viruses. This review describes the development of new Δγ(1)34.5 vectors, through serial passage selection and direct viral genome engineering, which demonstrate selective PKR evasion in targeted cells and improved viral replication without restoring neurovirulence.

HIV-1(89.6) Gag expressed from a replication competent HSV-1 vector elicits persistent cellular immune responses in mice.

We have constructed a replication competent, gamma(1)34.5-deleted herpes simplex virus type-1 (HSV-1) vector (J200) that expresses the gag gene from human immunodeficiency virus type-1, primary isolate 89.6 (HIV-1(89.6)), as a candidate vaccine for HIV-1. J200 replicates in vitro, resulting in abundant Gag protein production and accumulation in the extracellular media. Immunization of Balb/c mice with a single intraperitoneal injection of J200 elicited strong Gag-specific CD8 responses, as measured by intracellular IFN-gamma staining and flow cytometry analysis. Responses were highest between 6 weeks and 4 months, but persisted at 9 months post-immunization, the last time-point evaluated. These data highlight the potential utility of neuroattenuated, replication competent HSV-1 vectors for delivery of HIV-1 immunogens.

Oncolytic HSV-1 for the treatment of brain tumours.

The prognosis for patients diagnosed with malignant glioma, the most common primary tumour of the central nervous system, remains poor despite decades of research and advances in surgery, radiation and chemotherapy. The development of new approaches for the treatment of these tumours has led to the emergence of oncolytic viral therapy, with the use of conditionally replicating viruses, as a potential new intervention. Herpes simplex virus type 1 has emerged as the leading candidate oncolytic virus, with six different trials either completed or underway for patients with malignant glioma. In this review, the background of this approach will be discussed, followed by a discussion of the clinical trials, as well as potential directions for future trials.

Serial passage through human glioma xenografts selects for a Deltagamma134.5 herpes simplex virus type 1 mutant that exhibits decreased neurotoxicity and prolongs survival of mice with experimental brain tumors.

Previous studies have described in vitro serial passage of a Deltagamma(1)34.5 herpes simplex virus type 1 (HSV-1) strain in SK-N-SH neuroblastoma cells and selection of mutants that have acquired the ability to infect and replicate in this previously nonpermissive cell line. Here we describe the selection of a mutant HSV-1 strain by in vivo serial passage, which prolongs survival in two separate experimental murine brain tumor models. Two conditionally replication-competent Deltagamma(1)34.5 viruses, M002, which expresses murine interleukin-12, and its parent virus, R3659, were serially passaged within human malignant glioma D54-MG cell lines in vitro or flank tumor xenografts in vivo. The major findings are (i) viruses passaged in vivo demonstrate decreased neurovirulence, whereas those passaged in vitro demonstrate a partial recovery of the neurovirulence associated with HSV-1; and (ii) vvD54-M002, the virus selected after in vivo serial passage of M002 in D54-MG tumors, improves survival in two independent murine brain tumor models compared to the parent (unpassaged) M002. Additionally, in vitro-passaged, but not in vivo-passaged, M002 displayed changes in the protein synthesis profile in previously nonpermissive cell lines, as well as early U(S)11 transcription. Thus, a mutant HSV-1 strain expressing a foreign gene can be selected for enhanced antitumor efficacy via in vivo serial passage within flank D54-MG tumor xenografts. The enhanced antitumor efficacy of vvD54-M002 is not due to restoration of protein synthesis or early U(S)11 expression. This finding emphasizes the contribution of the in vivo tumor environment for selecting novel oncolytic HSV specifically adapted for tumor cell destruction in vivo.

Genetically engineered herpes simplex viruses that express IL-12 or GM-CSF as vaccine candidates.

We are using genetically modified, conditionally replicating herpes simplex virus (HSV) that express either interleukin (IL)-12 or granulocyte macrophage-colony stimulating factor (GM-CSF) as live, attenuated vaccine candidates for protection against HSV infection and/or disease. We report the following: (1) animals previously vaccinated with these candidate vaccines exhibited dose-dependent protection after intranasal, intraperitoneal or intracranial challenge with the highly virulent E377-MB wild-type HSV-1; (2) the IL-12 expressing virus (M002) consistently conferred protection at lower immunization doses than GM-CSF expressing virus (M004); (3) between 80 and 100% protection from E377-MB challenge was conferred after intramuscular immunization of mice with any of the three Deltagamma1 34.5 HSV, as opposed to 50% protection elicited after immunization with wild-type HSV-1 (F); and (4) latent virus was not detected at a higher rate in animals immunized and subsequently challenged with E377-MB than in immunized animals alone. These data suggest that conditionally replicating, cytokine-expressing HSV are able to elicit protective immune responses while retaining safety in an experimental murine model.