Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique.

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

Tumor-specific polycistronic miRNA delivered by engineered exosomes for the treatment of glioblastoma.

BACKGROUND: Glioblastoma (GBM) has poor prognosis due to ineffective agents and poor delivery methods. MicroRNAs (miRs) have been explored as novel therapeutics for GBM, but the optimal miRs and the ideal delivery strategy remain unresolved. In this study, we sought to identify the most effective pan-subtype anti-GBM miRs and to develop an improved delivery system for these miRs. METHODS: We conducted an unbiased screen of over 600 miRs against 7 glioma stem cell (GSC) lines representing all GBM subtypes to identify a set of pan-subtype-specific anti-GBM miRs and then used available TCGA GBM patient outcomes and miR expression data to hone in on miRs that were most likely to be clinically effective. To enhance delivery and expression of the miRs, we generated a polycistronic plasmid encoding 3 miRs (pPolymiR) and used HEK293T cells as biofactories to package pPolymiR into engineered exosomes (eExos) that incorporate viral proteins (Gag/VSVg) in their structure (eExos+pPolymiR) to enhance function. RESULTS: Our stepwise screen identified miR-124-2, miR-135a-2, and let-7i as the most effective miRs across all GBM subtypes with clinical relevance. Delivery of eExos+pPolymiR resulted in high expression of all 3 miRs in GSCs, and significantly decreased GSC proliferation in vitro. eExos+pPolymiR prolonged survival of GSC-bearing mice in vivo when compared with eExos carrying each of the miRs individually or as a cocktail. CONCLUSION: eExos+pPolymiR, which includes a pan-subtype anti-glioma-specific miR combination encoded in a polycistronic plasmid and a novel exosome delivery platform, represents a new and potentially powerful anti-GBM therapeutic.

Characteristics of pediatric COVID-19 infections and the impact of influenza and COVID-19 vaccinations during the first two years of the pandemic.

The data regarding the demographics of SARS-CoV-2 in the pediatric population has been published based on several single-center experiences or on metanalyses over short time frames. This article reports data on the demographics of pediatric patients with COVID-19 on a global scale using the TriNetX COVID-19 Research Network. In addition, we examined the risk of COVID-19 infection in relation to the body mass index (BMI) category and the protective value of influenza and COVID-19 immunization against COVID-19 infection. The incidence of COVID-19 infection was higher in the younger age group (≤6 years old), but no gender differences. The incidence of COVID-19 infection was higher among African Americans/Black race (28.57%) White race (27.10%), and obese patients; across all age groups, all genders, all races, and ethnicities (p < 0.0001). The incidence of MIS-C was also higher in patients with obesity (OR 1.71, CI 1.36-2.14). We found that the patients who were neither vaccinated for COVID-19 nor influenza within one year before their COVID-19 diagnoses compared to those who received influenza vaccine only, had significantly higher odds for hospitalization (OR 1.19, CI 1.18-1.21), development of MIS-C (OR 1.52, CI 1.32-1.74), and more importantly mortality (OR 1.47, CI 1.26-1.71). In addition, those patients who were neither vaccinated for COVID-19 nor influenza within one year before their COVID-19 diagnoses, compared to those who received at least one dose of COVID-19 vaccine, had significantly higher odds for hospitalization (OR 1.11, CI 1.04-1.19). However, those patients who did not receive the influenza vaccine within one year before their COVID-19 diagnoses nor received the COVID-19 vaccine had much higher odds for hospitalization (OR 1.46, CI 1.41-1.51), MIS-C (OR 3.72, CI 2.11-6.56), and mortality compared to those who received both vaccinations (OR 13.55, CI 1.91-9.62). Using the multiplicative interaction scale, we found a positive interaction between the COVID-19 vaccine and the influenza vaccine; they both combined have a larger effect than each separately. Our study is the largest of its kind (to date) examining the global demographic of the pandemic and the first of a kind to find a link between influenza vaccine and COVID-19-related hospitalization, MIS-C, and mortality in the pediatric population.

Measuring Nursing Student Development Through Computer-Based Simulation Activities.

BACKGROUND: New nurse attrition jeopardizes patient safety, devastates career plans, and negatively impacts costs to hospitals and patients. Employers and educators recognize the widening transition-to-practice gap in meeting expectations for practice-ready, resilient nurses. PURPOSE: The purpose of this study was to test the use of computer-based simulation activities (CBSAs) to measure processes in Aller's Development of Decision-Making and Self-Efficacy Model (ADD-SEM). METHODS: BSN students (N = 50) in a multisite, cross-sectional study used CBSAs to provide data on decision making (Virtual Patient Lasater's Clinical Judgment Rubric), psychological capital (Nursing Anxiety and Self-Confidence in Decision-Making Scale©), and reflection. RESULTS: Repeated-measures analysis of variance and reflexive thematic analysis revealed that decision-making (DM) scores were highest in noticing and lowest in interpreting with significant differences in cohorts (DM) (juniors: P < .001; seniors: P = .013) and self-confidence/anxiety ( P < .001). CONCLUSION: The CBSAs are an effective means of measuring student development of DM and psychological capital needed to retain resilient nurses.

An immune-competent, replication-permissive Syrian Hamster glioma model for evaluating Delta-24-RGD oncolytic adenovirus.

BACKGROUND: Oncolytic adenoviruses are promising new treatments against solid tumors, particularly for glioblastoma (GBM), and preclinical models are required to evaluate the mechanisms of efficacy. However, due to the species selectivity of adenovirus, there is currently no single animal model that supports viral replication, tumor oncolysis, and a virus-mediated immune response. To address this gap, we took advantage of the Syrian hamster to develop the first intracranial glioma model that is both adenovirus replication-permissive and immunocompetent. METHODS: We generated hamster glioma stem-like cells (hamGSCs) by transforming hamster neural stem cells with hTERT, simian virus 40 large T antigen, and h-RasV12. Using a guide-screw system, we generated an intracranial tumor model in the hamster. The efficacy of the oncolytic adenovirus Delta-24-RGD was assessed by survival studies, and tumor-infiltrating lymphocytes (TILs) were evaluated by flow cytometry. RESULTS: In vitro, hamGSCs supported viral replication and were susceptible to Delta-24-RGD mediated cell death. In vivo, hamGSCs consistently developed into highly proliferative tumors resembling high-grade glioma. Flow cytometric analysis of hamster gliomas revealed significantly increased T-cell infiltration in Delta-24-RGD infected tumors, indicative of immune activation. Treating tumor-bearing hamsters with Delta-24-RGD led to significantly increased survival compared to hamsters treated with phosphate buffered saline (PBS). CONCLUSIONS: This adenovirus-permissive, immunocompetent hamster glioma model overcomes the limitations of previous model systems and provides a novel platform to study the interactions between tumor cells, the host immune system, and oncolytic adenoviral therapy; understanding of which will be critical to implementing oncolytic adenovirus in the clinic.

RNAi technology targeting the FGFR3-TACC3 fusion breakpoint: an opportunity for precision medicine.

BACKGROUND: Fusion genes form as a result of abnormal chromosomal rearrangements linking previously separate genes into one transcript. The FGFR3-TACC3 fusion gene (F3-T3) has been shown to drive gliomagenesis in glioblastoma (GBM), a cancer that is notoriously resistant to therapy. However, successful targeting of F3-T3 via small molecular inhibitors has not revealed robust therapeutic responses, and specific targeting of F3-T3 has not been achieved heretofore. Here, we demonstrate that depleting F3-T3 using custom siRNA to the fusion breakpoint junction results in successful inhibition of F3-T3+ GBMs, and that exosomes can successfully deliver these siRNAs. METHODS: We engineered 10 unique siRNAs (iF3T3) that specifically spanned the most common F3-T3 breakpoint with varying degrees of overlap, and assayed depletion by qPCR and immunoblotting. Cell viability assays were performed. Mesenchymal stem cell-derived exosomes (UC-MSC) were electroporated with iF3T3, added to cells, and F3-T3 depletion measured by qPCR. RESULTS: We verified that depleting F3-T3 using shRNA to FGFR3 resulted in decreased cell viability and improved survival in glioma-bearing mice. We then demonstrated that 7/10 iF3T3 depleted F3-T3, and importantly, did not affect levels of wild-type (WT) FGFR3 or TACC3. iF3T3 decreased cell viability in both F3T3+ GBM and bladder cancer cell lines. UC-MSC exosomes successfully delivered iF3T3 in vitro, resulting in F3-T3 depletion. CONCLUSION: Targeting F3-T3 using siRNAs specific to the fusion breakpoint is capable of eradicating F3T3+ cancers without toxicity related to inhibition of WT FGFR3 or TACC3, and UC-MSC exosomes may be a plausible vehicle to deliver iF3T3.

Selective bladder denervation for overactive bladder (OAB) syndrome: From concept to healing outcomes using the ovine model.

AIMS: We evaluated a Selective Bladder Denervation (SBD) device, which uses radiofrequency ablation, for the treatment of overactive bladder syndrome in terms of its nerve denervation, ablation characteristics, and post-treatment healing. METHODS: Using the SBD device, eight fresh extirpated ovine bladder trigones were treated (90°C set point for 60 s) and nitroblue tetrazolium viability stained to characterize the ablation. In addition, 12 trigones were treated in vivo with three adjacent ablations and divided into survival cohorts: Day 7, Day 30, and Day 90 to assess the ablations and their associated healing. RESULTS: The ex vivo single trigone ablations had a 7.9 ± 0.9 mm width and 5.7 ± 1.0 mm thickness that involved the submucosa, detrusor muscle, adventitia, and vagina. Microscopic viability staining confirmed complete nerve necrosis within the targeted tissue. The in vivo Day 7 trigones supported the ex vivo ablation characteristics and showed up to minimal inflammation, granulation tissue, and collagen fibrosis. Day 30 trigones had essentially absent inflammation and granulation tissue with evolving collagen fibrosis at the ablation's periphery. Day 90 trigones had essentially absent acute inflammation, minimal chronic inflammation, essentially absent granulation tissue, and up to mild collagen fibrosis. No ureteral/urethral alterations, vesico-vaginal fistulas, or other complications were identified. CONCLUSIONS: The SBD device provided a targeted trigone ablation with resultant denervation. The tissue healing timeline followed that expected for a hyperthermic ablation and was characterized by a fibroproliferative healing response with limited inflammation and granulation tissue. The ablations did not impact the overlying bladder mucosal surface.

Exosomes from Glioma-Associated Mesenchymal Stem Cells Increase the Tumorigenicity of Glioma Stem-like Cells via Transfer of miR-1587.

Tumor-stromal communications impact tumorigenesis in ways that are incompletely understood. Here, we show that glioma-associated human mesenchymal stem cells (GA-hMSC), a newly identified stromal component of glioblastoma, release exosomes that increase the proliferation and clonogenicity of tumor-initiating glioma stem-like cells (GSC). This event leads to a significantly greater tumor burden and decreased host survival compared with untreated GSCs in orthotopic xenografts. Analysis of the exosomal content identified miR-1587 as a mediator of the exosomal effects on GSCs, in part via downregulation of the tumor-suppressive nuclear receptor corepressor NCOR1. Our results illuminate the tumor-supporting role for GA-hMSCs by identifying GA-hMSC-derived exosomes in the intercellular transfer of specific miRNA that enhance the aggressiveness of glioblastoma. Cancer Res; 77(21); 5808-19. ©2017 AACR.

IGFBP2 Activates the NF-κB Pathway to Drive Epithelial-Mesenchymal Transition and Invasive Character in Pancreatic Ductal Adenocarcinoma.

The molecular basis underlying the particularly aggressive nature of pancreatic ductal adenocarcinoma (PDAC) still remains unclear. Here we report evidence that the insulin-like growth factor-binding protein IGFBP2 acts as a potent oncogene to drive its extremely malignant character. We found that elevated IGFBP2 expression in primary tumors was associated with lymph node metastasis and shorter survival in patients with PDAC. Enforced expression of IGFBP2 promoted invasion and metastasis of PDAC cells in vitro and in vivo by inducing NF-κB-dependent epithelial-mesenchymal transition (EMT). Mechanistic investigations revealed that IGFBP2 induced the nuclear translocation and phosphorylation of the p65 NF-κB subunit through the PI3K/Akt/IKKß pathway. Conversely, enforced expression of PTEN blunted this signaling pathway and restored an epithelial phenotype to PDAC cells in the presence of overexpressed IGFBP2. Overall, our results identify IGFBP2 as a pivotal regulator of an EMT axis in PDAC, the activation of which is sufficient to confer the characteristically aggressive clinical features of this disease. Cancer Res; 76(22); 6543-54. ©2016 AACR.

Glioma progression is mediated by an addiction to aberrant IGFBP2 expression and can be blocked using anti-IGFBP2 strategies.

Insulin-like growth factor binding protein 2 (IGFBP2) overexpression is common in high-grade glioma and is both a strong biomarker of aggressive behaviour and a well-documented prognostic factor. IGFBP2 is a member of the secreted IGFBP family that functions by interacting with circulating IGFs to modulate IGF-mediated signalling. This traditional view of IGFBP2 activities has been challenged by the recognition of the diverse functions and cellular locations of members of the IGFBP family. IGFBP2 has been previously established as a driver of glioma progression to a higher grade. In this study, we sought to determine whether IGFBP2-overexpressing tumours are dependent on continued oncogene expression and whether IGFBP2 is a viable therapeutic target in glioma. We took advantage of the well-characterized RCAS/Ntv-a mouse model to create a doxycycline-inducible IGFBP2 model of glioma and demonstrated that the temporal expression of IGFBP2 has dramatic impacts on tumour progression and survival. Further, we demonstrated that IGFBP2-driven tumours are dependent on the continued expression of IGFBP2, as withdrawal of this oncogenic signal led to a significant decrease in tumour progression and prolonged survival. Inhibition of IGFBP2 also impaired tumour cell spread. To assess a therapeutically relevant inhibition strategy, we evaluated a neutralizing antibody against IGFBP2 and demonstrated that it impaired downstream IGFBP2-mediated oncogenic signalling pathways. The studies presented here indicate that IGFBP2 not only is a driver of glioma progression and a prognostic factor but is also required for tumour maintenance and thus represents a viable therapeutic target in the treatment of glioma. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Driver or passenger effects of augmented c-Myc and Cdc20 in gliomagenesis.

PURPOSE: Cdc20 and c-Myc are commonly overexpressed in a broad spectrum of cancers, including glioblastoma (GBM). Despite this clear association, whether c-Myc and Cdc20 overexpression is a driver or passenger event in gliomagenesis remains unclear. RESULTS: Both c-Myc and Cdc20 induced the proliferation of primary glial progenitor cells. c-Myc also promoted the formation of soft agar anchorage-independent colonies. In the RCAS/Ntv-a glia-specific transgenic mouse model, c-Myc increased the GBM incidence from 19.1% to 47.4% by 12 weeks of age when combined with kRas and Akt3 in Ntv-a INK4a-ARF (also known as CDKN2A)-null mice. In contrast, Cdc20 decreased the GBM incidence from 19.1% to 9.1%. Moreover, cell differentiation was modulated by c-Myc in kRas/Akt3-induced GBM on the basis of Nestin/GFAP expression (glial progenitor cell differentiation), while Cdc20 had no effect on primary glial progenitor cell differentiation. MATERIALS AND METHODS: We used glial progenitor cells from Ntv-a newborn mice to evaluate the role of c-Myc and Cdc20 in the proliferation and transformation of GBM in vitro and in vivo. We further determined whether c-Myc and Cdc20 have a driver or passenger role in GBM development using kRas/Akt3 signals in a RCAS/Ntv-a mouse model. CONCLUSIONS: These results suggest that the driver or passenger of oncogene signaling is dependent on cellular status. c-Myc is a driver when combined with kRas/Akt3 oncogenic signals in gliomagenesis, whereas Cdc20 overexpression is a passenger. Inhibition of cell differentiation of c-Myc may be a target for anti-glioma therapy.

Building a funded research program in cancer health disparities: considerations for young investigators.

A workshop entitled "Building a funded research program in cancer health disparities" was held at the 38th Annual American Society of Preventive Oncology (ASPO) Meeting. Organized by the Junior Members Interest Group, the session addressed topics relevant to career development for cancer disparities investigators. Such considerations include the development of research programs on a backdrop of existing multi- and transdisciplinary teams, recognizing opportunities for advancing their research, given the growth of consortia-related research, and development of effective community-based partnerships. Key strategies for developing a sustainable career in cancer health disparities in the current environment include the need to effectively engage with communities, appreciate the value of team science and develop cross-discipline collaborations, and navigate the use and utility of consortia for disparities research. Academic considerations related to earning tenure and promotion that may be faced by the junior investigator in cancer health disparities were also discussed. This report may serve to both educate and provide lessons for early-stage investigators who wish to tackle complex scientific questions while developing their careers in cancer health disparities.

Human papillomavirus vaccine awareness, uptake, and parental and health care provider communication among 11- to 18-year-old adolescents in a rural Appalachian Ohio county in the United States.

PURPOSE: This study examined human papillomavirus (HPV) vaccine awareness and uptake, and communication with a parent and/or a health care provider among 11- to 18-year-old male and female adolescents in an Appalachian Ohio county. METHODS: Five questions regarding the HPV vaccine were added to the 2012 Youth Risk Behavior Surveillance System (YRBSS) surveys administered to middle and high school students in the county. The YRBSS surveys are school-based, anonymous, and voluntary. The questions added were about vaccine awareness and uptake, and communication with a parent or health care provider about the vaccine. RESULTS: Of the 1,299 participants, 51.9% were male and 90.3% were white. Overall, 49.2%, 23.5%, 19.2%, and 24.6%, respectively, reported vaccine awareness, uptake of at least 1 dose of the HPV vaccine, communication with a parent, and communication with a health care provider. Females and adolescents ≥ 15 years were significantly more likely to report awareness, uptake, and parental and provider communication than males and adolescents ≤ 14 years. Adolescents receiving any dose of the vaccine were significantly more likely to have had a parent (OR: 3.74; 95% CI: 2.30-6.06) or a health care provider (OR: 10.91; 95% CI: 6.42-18.6) discuss the vaccine than those who had not received any dose. CONCLUSIONS: Despite the strong link between parental and health care provider communication and HPV vaccine uptake, the levels of communication remain low in this Appalachian population. These findings suggest the need for public health education programs targeting the health care providers, the parents, and the adolescents to improve awareness, knowledge, and HPV vaccine uptake.

Occlusion of canine atrial appendage using an expandable silicone band.

OBJECTIVE: Because the left atrial appendage is thought to be a major source of stroke in patients with atrial fibrillation, a new device and technique were developed for thoracoscopic isolation of the left atrial appendage. METHODS: The left and right atrial appendages were approached from an 11.5-mm port in the left thorax in 15 canines. With an atraumatic grasper for appendage positioning, expandable silicone bands covered with polyester fabric were placed at the base of the left and right atrial appendages. The location of the bands was marked with radiopaque clips to assess migration, and radiopaque dye was injected to confirm occlusion. The animals were killed at 1 week (n = 3), 2 weeks (n = 6), or 12 weeks (n = 6). RESULTS: The bands were deployed on 30 appendages without complications. The appendages were 100% occluded, and there was no migration of any bands at death. There was no indication of bleeding, rupture, or systemic emboli in any of the 15 animals. Following complete occlusion, the appendages became necrotic and were replaced by scar tissue. The healed atrial surface was consistently smooth and devoid of thrombus. There was evidence of mild-to-moderate inflammation associated with a foreign body-type reaction to the fabric material but no pericarditis. CONCLUSIONS: Permanent occlusion of the left atrial appendage is feasible via a thoracoscopic epicardial approach with this novel silicone band. As the atrial tissue becomes necrotic, the silicone band continues to constrict, ensuring that the appendage remains isolated. Band occlusion of the left atrial appendage could potentially improve overall treatment outcome in patients at high risk of atrial thrombus formation.

Band occlusion of the atrial appendage.

OBJECTIVES: A new device has been developed for occlusion of the left atrial appendage (LAA). Previous investigations have been in the canine model. The canine atrial appendage does not vary in size and shape as much as the human. The goal of this study was to investigate band occlusion of right atrial appendages in the pig model, which are more broad based. METHODS: The right atrial appendages of six pigs were approached through a right thoracotomy. An expandable polyester fabric-covered silicone band was placed on the appendage. The animals were evaluated at the time of band placement with fluoroscopy, contrast injection, and in three animals, echocardiography. After one week, the animals were again evaluated with fluoroscopy. The animals were sacrificed at 12 weeks. RESULTS: Bands were placed without complication at the base of all six appendages. All appendages were effectively occluded and all bands remained at the appendage base. The healed atrial surface was consistently smooth and devoid of thrombus. CONCLUSIONS: Occlusion of a broad-based appendage as seen with human LAA is feasible with this novel band technique.

Multivariate association analysis of the components of metabolic syndrome from the Framingham Heart Study.

Metabolic syndrome, by definition, is the manifestation of multiple, correlated metabolic impairments. It is known to have both strong environmental and genetic contributions. However, isolating genetic variants predisposing to such a complex trait has limitations. Using pedigree data, when available, may well lead to increased ability to detect variants associated with such complex traits. The ability to incorporate multiple correlated traits into a joint analysis may also allow increased detection of associated genes. Therefore, to demonstrate the utility of both univariate and multivariate family-based association analysis and to identify possible genetic variants associated with metabolic syndrome, we performed a scan of the Affymetrix 50 k Human Gene Panel data using 1) each of the traits comprising metabolic syndrome: triglycerides, high-density lipoprotein, systolic blood pressure, diastolic blood pressure, blood glucose, and body mass index, and 2) a composite trait including all of the above, jointly. Two single-nucleotide polymorphisms within the cholesterol ester transfer protein (CETP) gene remained significant even after correcting for multiple testing in both the univariate (p < 5 x 10-7) and multivariate (p < 5 x 10-9) association analysis. Three genes met significance for multiple traits after correction for multiple testing in the univariate analysis, while five genes remained significant in the multivariate association. We conclude that while both univariate and multivariate family-based association analysis can identify genes of interest, our multivariate approach is less affected by multiple testing correction and yields more significant results.

No association between phosphatase and tensin homolog genetic polymorphisms and colon cancer.

AIM: To investigate the association between single nucleotide polymorphisms (SNPs) in the phosphatase and tensin homolog (PTEN) tumor suppressor gene and risk of colon cancer. METHODS: We utilized a population-based case-control study of incident colon cancer individuals (n = 421) and controls (n = 483) aged > or = 30 years to conduct a comprehensive tagSNP association analysis of the PTEN gene. RESULTS: None of the PTEN SNPs were statistically significantly associated with colon cancer when controlled for age, gender, and race, or when additionally adjusted for other known risk factors (P > 0.05). Haplotype analyses similarly showed no association between the PTEN gene and colon cancer. CONCLUSION: Our study does not support PTEN as a colon cancer susceptibility gene.

Effects of a DNA vaccine in an animal model of Alternaria alternata sensitivity.

Sensitivity to the fungus Alternaria is associated with asthma persistence and severity. Current therapeutic options for treating Alternaria-induced airway inflammation are limited. In this study, Brown Norway rats are used to study the effectiveness of a DNA-based vaccine delivered to the airway in attenuating the response to a major Alternaria allergen, rAlt a 2. Compared to untreated sensitized animals, or animals receiving an "out-of-frame" DNA-based vaccine, animals treated with "in-frame" DNA vaccine showed an attenuation in specific IgE antibody titers to rAlt a 2, an increase in IgG(2b) (a Th1 response), a reduction in spontaneous IL-13 release by peribronchial lymph node cell suspensions, and an attenuation in the decrease in total lung capacity 72 h post-allergen challenge. Further, histopathologic examination of the lung tissues revealed reduced pulmonary inflammation post-allergen challenge in the DNA-vaccine-treated compared to sensitized, untreated animals. We conclude that a DNA-based vaccine delivered to the airway significantly influences the immunologic, pulmonary physiologic, and histological alterations induced by challenge with a major Alternaria allergen, rAlt a 2, in sensitized animals.