Exosomes in Allergic Airway Diseases.

PURPOSE OF REVIEW: This review will cover what is known regarding exosomes and allergy, and furthermore discuss novel mechanism of exosome-mediated immune modulation and metabolic regulation via the transfer of mitochondria. RECENT FINDINGS: Exosomes are nano-sized extracellular vesicles (EVs) derived from the endosome that play a direct role in governing physiological and pathological conditions by transferring bioactive cargo such as proteins, enzymes, nucleic acids (miRNA, mRNA, DNA), and metabolites. Recent evidence suggest that exosomes may signal in autocrine but, most importantly, in paracrine and endocrine manner, being taken up by neighboring cells or carried to distant sites. Exosomes also mediate immunogenic responses, such as antigen presentation and inflammation. In asthma and allergy, exosomes facilitate cross-talk between immune and epithelial cells, and drive site-specific inflammation through the generation of pro-inflammatory mediators like leukotrienes. Recent studies suggest that myeloid cell-generated exosomes transfer mitochondria to lymphocytes. Exosomes are nano-sized mediators of the immune system which can modulate responses through antigen presentation, and the transfer of pro- and anti-inflammatory mediators. In addition to conventional mechanisms of immune modulation, exosomes may act as a novel courier of functional mitochondria that is capable of modulating the recipient cells bioenergetics, resulting in altered cellular responses. The transfer of mitochondria and modulation of bioenergetics may result in immune activation or dampening depending on the context.

Exosomes in immunoregulation of chronic lung diseases.

Exosomes are nano-sized, membrane-bound vesicles released from cells that transport cargo including DNA, RNA, and proteins, between cells as a form of intercellular communication. In addition to their role in intercellular communication, exosomes are beginning to be appreciated as agents of immunoregulation that can modulate antigen presentation, immune activation, suppression, and surveillance. This article summarizes how these multifaceted functions of exosomes may promote development and/or progression of chronic inflammatory lung diseases including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. The potential of exosomes as a novel therapeutic is also discussed.

Refractory asthma: mechanisms, targets, and therapy.

Asthma is a common medical condition affecting 300 million people worldwide. Airway inflammation, smooth muscle bronchoconstriction leading to airflow obstruction, and mucous hypersecretion are clinical hallmarks of asthma. The NHLBI Expert Panel Report 3 recommends inhaled corticosteroids (ICS) for patients with moderate to severe persistent asthma. Inhaled corticosteroids (ICS) target gene transcription through their interactions with the glucocorticoid (GC) receptor (GR) at the glucocorticoid response element (GRE). The GC/GR complex enhances anti-inflammatory but inhibits pro-inflammatory mediator production. Classically, asthma has been described as a Th2-associated eosinophil-predominant disease, but recently alternative models have been described including a Th17-mediated neutrophil-predominant phenotype resulting in patients with more severe disease who may be less responsive to steroids. Additional mechanisms of steroid resistance include increased activity of GR phosphorylating kinases which modify the interactions of GR with transcription factors to inhibit the ability of GR to bind with GRE, leading to an increase in pro-inflammatory gene transcription. Oxidative stress also affects the balance between pro-inflammatory and anti-inflammatory gene transcription through the modification of transcription factors and cofactors (such as PI3K) leading to the inhibition of histone deacetylase 2. Continued investigations into the mechanisms behind glucocorticoid resistance will lead to novel treatments that improve control of severe refractory asthma.

A myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism.

Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here, we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165,000-fold enhanced lung targeting, compared with Ad5. In addition, myeloid cell-specific binding is preserved in single-cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest that endothelial transduction is achieved by leukocyte-mediated 'hand-off' of Ad.

Targeted tumor killing via an intracellular antibody against erbB-2.

Specific killing of erbB-2-overexpressing tumor cells can be achieved using expression of an intracellular antibody directed against the erbB-2 oncoprotein. We have developed a strategy using a recombinant adenovirus encoding an anti-erbB-2 single chain antibody to achieve targeted tumor cell killing in vivo and can show significantly prolonged survival of animals carrying a human ovarian carcinoma tumor burden within their peritoneal cavities. This strategy of gene therapy for ovarian carcinoma offers the potential to achieve highly specific, targeted killing of human tumor cells and thus establishes the rationale to undertake human clinical trials on this basis.

Neoplastic reversion accomplished by high efficiency adenoviral-mediated delivery of an anti-ras ribozyme.

Strategies have been developed to abrogate the aberrant expression of dominant oncogenes as a means to accomplish targeted tumor eradication. We have demonstrated previously the utility of this approach using a hammerhead ribozyme designed to cleave the mutant sequence in codon 12 of the activated H-ras oncogene transcript. To develop this strategy into a practical means to approach malignant disease, methods must be developed to accomplish high efficiency delivery of the ribozyme to target neoplastic cells. To accomplish this, a recombinant adenovirus was designed that encoded a gene cassette for the H-ras ribozyme. By using this virus, it was possible to accomplish high efficiency reversion of the neoplastic phenotype in mutant H-ras expressing tumor cells without the need for any selection steps. The demonstration of the utility of adenoviral-mediated delivery of anticancer ribozymes will allow the practical development of gene therapy strategies on this basis.

Novel gene therapy strategy to accomplish growth factor modulation induces enhanced tumor cell chemosensitivity.

erbB-2 is a cell surface transmembrane glycoprotein which, when overexpressed, has been shown to be relevant to intrinsic tumor cell chemoresistance. Thus, strategies to down-regulate cell surface erbB-2 have resulted in enhanced tumor cell chemosensitivity. We have recently reported a gene therapy strategy to down-modulate erbB-2 expression using a plasmid construct encoding an intracellular single chain antibody. Therefore, we now demonstrate enhanced chemosensitivity to cis-diamminedichloroplatinum in erbB-2 overexpressing tumor cells and a model system of stable clones using an intracellular single chain antibody. These findings are consistent with the hypothesis that erbB-2 plays a role in tumor cell chemoresistance. In addition, these findings represent a novel gene therapy approach to overcome erbB-2-mediated tumor cell chemoresistance.

The level of erbB2 expression predicts sensitivity to the cytotoxic effects of an intracellular anti-erbB2 sFv.

We have previously demonstrated that an intracellular antibody (sFv) directed against erbB2 can achieve a specific cytotoxicity in erbB2 overexpressing cancer cells of varying histogenesis. In order to further delineate the mechanistic basis of the induced apoptosis, transient and stable cotransfections were performed. Transient cotransfection of erbB2 mutant and chimeric molecules demonstrated that the cytoplasmic domain of erbB2, or the homologous cytoplasmic domain of the epidermal growth factor receptor, is required for apoptosis induction. These results were confirmed in assays utilizing differential derivation of stable clones. To examine the effects of varying ratios of the anti-erbB2 sFv and its target erbB2 we performed additional cotransfection experiments in erbB2 negative target cells. When erbB2 levels are held constant, observed cytotoxicity is proportional to the amount of sFv added. In addition, when sFv levels are held constant, increasing levels of cotransfected erbB2 can overcome the apoptotic response. These results indicate that a minimal threshold level of the sFv and its target are required to induce cytotoxicity. To examine this phenomenon in an erbB2 positive cell line, SKOV3 ovarian carcinoma cells were utilized to derive a stable clone expressing low levels of sFv. When this cell line was compared to the parental SKOV3 cell line, it was shown that less exogenous sFv was needed to induce cytotoxicity in the clone already expressing low levels of sFv, indicating that endogenous and exogenous levels of sFv are additive. In summary, the results presented here indicate that the carboxy-terminus of the intracellular domain of the erbB2 molecule is involved in the induction of apoptosis. Furthermore, the expression levels of the sFv and its target protein need to overcome a threshold level in order to achieve a cytotoxic response.

A novel gene therapy strategy for elimination of prostate carcinoma cells from human bone marrow.

We report a novel means to purge bone marrow of a specific subset of prostate carcinoma cells based on transductional and genetic selectivity. Using both adenovirus-polylysine-DNA complexes and E1A/B-deleted replication-deficient adenoviruses, we have demonstrated a transductional preference of these vectors for the prostate carcinoma cell lines DU 145, LNCaP, and PC-3 over primary human bone marrow cells and the leukemia cell line KG-1. We have also shown a genetic selectivity of an anti-erbB-2 intracellular single-chain antibody (sFv) encoding adenovirus, Ad21, for the erbB-2-positive prostate carcinoma cell lines DU 145 and LNCaP. Delivery of Ad21 resulted in cytotoxicity to the DU 145 and LNCaP, but not PC-3, cell lines and reduced the clonogenic capacity of DU 145 cells cultured alone or mixed with various ratios of irradiated human bone marrow. Finally, quantitative, competitive reverse transcription polymerase chain reaction (QC-RT-PCR) analysis demonstrated that Ad21 could effectively reduce DU 145 and erbB-2-positive primary prostate tumor contamination in bone marrow cultures. Delivery of Ad21 had no effect on the ability of progenitor cells to form colonies. These results suggest that an anti-erbB-2 sFv-encoding adenoviral vector is efficacious for removal of erbB-2-positive prostate carcinoma cells from human bone marrow, and demonstrates a novel method for ex vivo genetic purge of malignant cells from bone marrow for autologous bone marrow transplantation (ABMT) therapy.

Intracellular antibody against erbB-2 mediates targeted tumor cell eradication by apoptosis.

Methods were developed to achieve targeted eradication of the erbB-2 oncoprotein using gene constructs encoding anti-erbB-2 intracellular single-chain antibodies. This method of genetic intervention caused a marked cytocidal effect in erbB-2-overexpressing human ovarian tumor cells. Evaluation of the mechanistic basis of this phenomenon demonstrated that programmed cell death had been induced. Significantly, no cytocidal effect was observed in non-erbB-2-overexpressing tumors. The induction of apoptosis could be shown to be secondary to the intracellular antibody-mediated ectopic localization of the erbB-2 oncoprotein. Thus, the strategy of selective oncogene "knock-out" using intracellular antibodies represents a novel anticancer gene therapy strategy that offers the potential to achieve highly specific, targeted eradication of human tumor cells.

Gene therapy and ovarian cancer: a review.

OBJECTIVE: To provide the obstetrician-gynecologist with the basic concepts of gene therapy, an overview of cancer gene therapy treatment strategies, and a summary of currently approved human gene therapy protocols for ovarian carcinoma. DATA SOURCES: A computerized search of articles published through December 1995 was performed on the MEDLINE data base. Additional sources were identified through cross-referencing. METHODS OF STUDY SELECTION: All identified references were reviewed with particular attention to their relevance to gene therapy for ovarian cancer. TABULATION, INTEGRATION, AND RESULTS: Each reference was reviewed to determine the relevant contribution to the fundamental science of gene therapy. Particular attention was paid to those studies that offered a rational strategy that might contribute to therapy for ovarian malignancy. CONCLUSION: Advances in molecular biology, immunology, and virology have enabled the concept of gene therapy for neoplastic disorders to become a reality. Given the lack of effective conventional therapy, those patients with recurrent or refractory ovarian cancer should be considered for currently approved investigational gene therapy protocols.

Eradication of c-erbB-2-Positive Ovarian Cancer Cells Mediated by Intracellular Expression of Anti-c-erbB-2 Antibody.

Overexpression of erbB-2 is important in the pathogenesis of a variety of human neoplasms. Overexpression of the erbB-2 gene product has been associated with poor clinical prognosis with respect to malignancies originating in the ovary, breast, gastrointestinal tract, salivary gland, and lung (1-4) and has led to the development of several therapeutic strategies to target tumor cells exhibiting increased surface levels of erbB-2. Monoclonal antibodies that exhibit high-affinity binding to the extracellular domains of the erbB-2 protein have been developed (5,6). Several studies have demonstrated that a subset of these antibodies can elicit growth inhibition of erbB-2 overexpressing cells both in vitro and in vivo (5,6). Antitumor therapies directed at erbB-2 have also been developed utilizing targeted immunotoxins (7). Gene-therapy strategies such as antisense technology has been widely used in these areas of research to achieve selected knockout of genes both at transcriptional or posttranscriptional levels (8-10). Recombinant fusion proteins consisting of various bacterial toxins selectively targeted to the tumor by virtue of single-chain anti-erbB-2 antibody (sFv) moieties has also been utilized in this context (7).

Free radical-producing myeloid-derived regulatory cells: potent activators and suppressors of lung inflammation and airway hyperresponsiveness.

Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of inducible nitric oxide synthase (iNOS), arginase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of their infiltration into the lung. The nitric oxide-producing cells were Ly-6C(+)Ly-6G(-) and they downmodulated T-cell activation, recruited T(reg) cells, and dramatically downregulated antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C(-)Ly-6G(+) and they expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a superoxide-dependent fashion. A smaller population of Ly-6C(+)Ly-6G(+) cells also suppressed T-cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells represent important targets for asthma therapy.

Regulation of casein kinase 2 by direct interaction with cell surface receptor CD5.

The transmembrane protein CD5, expressed on all T cells and the B1 subset of B cells, modulates antigen receptor-mediated activation. We used the yeast two-hybrid system to identify proteins that interact with its cytoplasmic domain and play a role in CD5 proximal signaling events. We found that the beta subunit of the serine/threonine kinase casein kinase 2 (CK2) interacts specifically with the cytoplasmic domain of CD5. Co-immunoprecipitation experiments showed activation-independent association of CK2 with CD5 in human and murine B and T cell lines and murine splenocytes. The interaction of CK2 holoenzyme with CD5 is mediated by the amino terminus of the regulatory subunit beta. CK2 binds and phosphorylates CD5 at the CK2 motifs flanked by Ser459 and Ser461. Cross-linking of CD5 leads to the activation of CD5-associated CK2 in a murine B-lymphoma cell line and a human T-leukemia cell line and is independent of net recruitment of CK2 to CD5. In contrast, CK2 is not activated following cross-linking of the B cell receptor complex or the T cell receptor complex. This direct regulation of CK2 by a cell surface receptor provides a novel pathway for control of cell activation that could play a significant role in regulation of CD5-dependent antigen receptor activation in T and B cells.

erbB-2 knockout employing an intracellular single-chain antibody (sFv) accomplishes specific toxicity in erbB-2-expressing lung cancer cells.

erbB-2 is known to be overexpressed in several human malignancies including lung cancer. Because of its role in neoplastic transformation as well as its association with poor prognosis, this oncogene has been targeted through various anti-cancer methodologies. In this regard, we have recently demonstrated that erbB-2-overexpressing ovarian tumor cell lines transfected with an endoplasmic reticulum form of an anti-erbB-2 single-chain antibody undergo a specific cytotoxicity through the induction of apoptosis. Since certain forms of lung cancer are also associated with overexpression of erbB-2, we evaluated the use of this novel therapeutic in this context. For these studies, several human lung adenocarcinoma cell lines were stably and transiently transfected with the anti-erbB-2 sFv gene. We demonstrate here that the anti-erbB-2 sFv can cause specific cytotoxicity in lung cancer cells. As a first step toward clinical translation of this strategy, we constructed a replication-deficient recombinant adenoviral vector expressing the anti-erbB-2 sFv construct. We further demonstrate that our anti-erbB-2 sFv-encoding adenoviral vector can accomplish high levels of cytotoxicity in lung cancer cells. Based on these results, it is proposed that this strategy of oncoprotein ablation may have use in the treatment of some forms of human lung cancer.

An intracellular anti-erbB-2 single-chain antibody is specifically cytotoxic to human breast carcinoma cells overexpressing erbB-2.

We previously demonstrated that delivery of a gene encoding an anti-erbB-2 intracellular single-chain antibody (sFv) resulted in down-regulation of cell surface erbB-2 levels and induction of apoptosis in erbB-2 overexpressing ovarian cancer cells. Based upon these findings, we hypothesized that human breast carcinomas overexpressing erbB-2 would be similarly affected by this genetic intervention. We evaluated the phenotypic effects resulting from intracellular expression of the anti-erbB-2 sFv on the human breast cancer cell lines MDA-MB-361, SK-BR-3, BT-474, MCF-7 and MDA-MB-231. Recombinant adenoviruses encoding either a reporter gene (AdCMVLacZ) or the endoplasmic reticulum (ER) directed anti-erbB-2 sFv (Ad21) were delivered to various breast cancer cell lines. Cell viability was determined by a proliferation assay and fluorescent microscopy allowed visualization of apoptotic cells. An erbB-2 ELISA quantified the endogenous erbB-2 levels of each cell line. The anti-erbB-2 sFv-encoding-adenovirus, Ad21, but not the beta-galactosidase encoding adenovirus, AdCMVLacZ, was cytotoxic to > 95% of the tumor cells in the MDA-MB-361 and SK-BR-3 lines, and > 60% of the tumor cells in the BT-474 line. In marked contrast, the MCF-7 and MDA-MB-231 cell lines showed no change in the rate of cell proliferation following this treatment. The cytotoxic effects generated in the first three lines were a consequence of the induction of apoptosis by the anti-erbB-2 sFv. An ELISA specific for erbB-2 showed that the breast cancer cell lines most susceptible to the anti-erbB-2 sFv, MDA-MB-361, SK-BR-3 and BT-474, overexpressed the erbB-2 protein while the cell lines demonstrating no response to the anti-erbB-2 sFv, MCF-7 and MDA-MB-231, expressed the lowest levels of erbB-2. These results demonstrate that targeted killing of erbB-2 overexpressing cells via intracellular knockout can be accomplished in the context of breast carcinoma. Furthermore, erbB-2 levels in breast tumor cells may be predictive of their sensitivity to sFv-mediated killing. The ability to accomplish selective cytotoxicity of breast cancer cell lines overexpressing the erbB-2 tumor marker should allow for derivation of clinical gene therapy strategies for breast cancer utilizing this approach.

Transductional efficacy and safety of an intraperitoneally delivered adenovirus encoding an anti-erbB-2 intracellular single-chain antibody for ovarian cancer gene therapy.

We have previously shown that adenoviral-mediated delivery of an anti-erbB-2 intracellular single-chain antibody (sFv) causes specific cytotoxicy in erbB-2-overexpressing ovarian carcinoma cells. Furthermore, intraperitoneal delivery of the anti-erbB-2 sFv enhances survival and reduces tumor burden in a xenograft model of human ovarian carcinoma in SCID mice. These findings have led to an RAC-approved Phase I clinical trial for patients with ovarian cancer. In this report, we show that expression of the anti-erbB-2 sFv could be readily detected in target tumor cells by in situ hybridization methodology. PCR analysis of DNA extracted from various murine tissues demonstrated that the anti-erbB-2 sFv remained localized to the peritoneum. Delivery of the sFv to the non-erbB-2-overexpressing REN mesothelial and Hep G2 hepatocellular carcinoma cell lines was not deleterious to either one, affirming the tumor specificity of this gene therapy strategy. In addition, histopathological analysis of various tissues showed that adenoviral-mediated delivery of the anti-erbB-2 sFv to immunocompetent mice with either primary exposure or previous vector challenge at different doses produced no abnormal changes when compared to untreated animals. These findings suggest that adenoviral-mediated delivery of the anti-erbB-2 sFv in a human context can be effectively assayed, is potentially free of vector-associated toxicity, and retains biologic utility based on tumor specificity.

Targeted eradication of ovarian cancer mediated by intracellular expression of anti-erbB-2 single-chain antibody.

OBJECTIVE: Overexpression of the tyrosine kinase receptor erbB-2 is important in the pathogenesis of a variety of neoplasms including ovarian cancer. As a strategy to selectively eradicate erbB-2-overexpressing tumor cells, an anti-erbB-2 single-chain immunoglobin (sFv) gene was constructed to direct expression of intracellular anti-erbB-2 antibody. The purpose of this study is to establish the antitumorigenicity of this strategy in in vitro and in vivo models. METHODS: An anti-erbB-2 sFv construct containing an endoplasmic reticulum (ER)-directed leader sequence was transiently expressed in the human ovarian carcinoma cell line SKOV3 using the adenovirus-polylysine vector. SKOV3 cells transfected with a non-ER form of an anti-erbB-2 sFv construct or an irrelevant plasmid DNA served as controls. Antitumorigenicity, as measured by anchorage-independent growth in soft agar and subcutaneous (sc) tumor formation, was analyzed. The ability to achieve a biological effect with relevant sFv in murine orthotopic xenograft models and in primary human ovarian cancer cells was also evaluated. RESULTS: The ER form of anti-erbB-2 sFv was found to exert a marked antineoplastic effect on the SKOV3 cell line resulting in an arrest of anchorage-independent growth. A significant increase in sc tumor volume was noted in animals challenged with control constructs. In marked contrast, complete tumor eradication was noted at necropsy 80 days after sc transplantation in the group challenged with the ER-directed anti-erbB-2 sFv gene. Intraperitoneal treatment of malignant ascites in human tumor xenograft models with the ER form of the anti-erbB-2 sFv gene resulted in profound downregulation of cell surface erbB-2 in retrieved ovarian cancer cells. The ER-directed anti-erbB-2 sFv also elicited a significant cytotoxic effect in transfected primary ovarian cancer cells obtained from a patient with malignant ascites. CONCLUSION: The ability to selectively "knock out" erbB-2 demonstrates that this strategy can induce a significant antineoplastic effect in ovarian cancer cells overexpressing this growth factor receptor. In addition, the ability to accomplish selective abrogation of erbB-2 expression in animal treatment models and to transfect and eradicate primary ovarian cancer cells justifies further investigation of this novel strategy in ovarian cancer patients.

Intracellular single-chain antibody directed against erbB2 down-regulates cell surface erbB2 and exhibits a selective anti-proliferative effect in erbB2 overexpressing cancer cell lines.

Overexpression of the tyrosine kinase receptor erbB2 is important in the pathogenesis of a variety of neoplasms. Based on this concept, targeted anti-cancer strategies have been designed to selectively eradicate erbB2 overexpressing tumor cells. These strategies have employed either monoclonal antibodies or antibody toxin molecules with specificity for the cell surface erbB2 protein. As an alternative strategy, anti-erbB2 single-chain immunoglobulin (sFv) genes were constructed to direct expression of intracellular anti-erbB2 antibodies. Expression of an endoplasmic reticulum (ER) form of the anti-erbB2 sFv resulted in a profound down-regulation of cell surface erbB2 in an erbB2 overexpressing ovarian carcinoma cell line. In addition, expression of the intracellular antibody resulted in marked inhibition of tumor cell proliferation. Whereas stable transfectants expressing the anti-erbB2 sFv could be dervied from non-erbB2 overexpressing cancer cell lines, expression of the intracellular antibody was incompatible with long-term survival of the erbB2 overpressing tumor cells. The ability to selectively 'knock-out' erbB2 demonstrates that cell surface localization of erbB2 is essential to its ability to induce aberrant cellular proliferation in tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)