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Objectives: Point-of-care ultrasound (POCUS) use is widespread in EDs and throughout those practising medicine. Between institutions and specialities, there is widespread variety and training. With this comes the risk of patient harm and backlash to a clinically useful modality. Our objective is to form a statement that encompasses current published and unpublished guidance for creating and maintaining robust POCUS programs in EDs., Methods: Emergency Medicine Ultrasound Group (EMUG) identified this gap and volunteers from the group undertook a literature search of current best practice and institution guidelines relating to POCUS programs. They contacted colleagues from other specialities to find and get access to other countries and colleges' POCUS guidelines. EMUGs regularly run discussion forums (Collab-labs) and points from these were considered. Recommendations were then formed from these and recurrent unpublished obstacles the group had encountered. The result was reviewed by clinical leaders in ultrasound and POCUS users in Australasia., Results: The recommendations were organised under five pillars: Infrastructure, Governance, Administration, Education and Quality., Conclusion: These recommendations complement existing guidelines and are not intended to replace them; however, we hope to promote discussion and provide reference support for those developing POCUS programs. Implementing a comprehensive and robust ED POCUS program will ensure safe, effective and standardised high-quality POCUS use, with the aim of improving patient care across Australia and New Zealand. Patient safety will be enhanced through effective risk management and quality assurance and there will be consistency in POCUS education, training and credentialing across institutions., (© 2024 The Author(s). Emergency Medicine Australasia published by John Wiley & Sons Australia, Ltd on behalf of Australasian College for Emergency Medicine.)

Objectives: Point-of-care ultrasound (POCUS) use is widespread in EDs and throughout those practising medicine. Between institutions and specialities, there is widespread variety and training. With this comes risk of patient harm and backlash to a clinically useful modality. Our objective is to form a statement that encompasses current published and unpublished guidance for creating and maintaining robust POCUS programs in EDs., Methods: Emergency Medicine Ultrasound Group (EMUG) identified this gap and volunteers from the group undertook a literature search of current best practice and institution guidelines relating to POCUS programs. They contacted colleagues from other specialities to find and get access to other countries and colleges' POCUS guidelines. EMUGs regularly run discussion forums (Collab-labs) and points from these were considered. Recommendations were then formed from these and recurrent unpublished obstacles the group had encountered. The result was reviewed by Clinical Leads in Ultrasound and POCUS users in Australasia., Results: The recommendations were organised under five pillars: Infrastructure, Governance, Administration, Education and Quality., Conclusion: These recommendations complement existing guidelines and are not intended to replace them; however, we hope to promote discussion and provide reference support for those developing POCUS programs. Implementing a comprehensive and robust ED POCUS program will ensure safe, effective, and standardised high-quality POCUS use, with the aim of improving patient care across Australia and New Zealand. Patient safety will be enhanced through effective risk management and quality assurance and there will be consistency in POCUS education, training and credentialing across institutions., (© 2024 The Author(s). Emergency Medicine Australasia published by John Wiley & Sons Australia, Ltd on behalf of Australasian College for Emergency Medicine.)

Primary progressive aphasia (PPA) is a neurodegenerative brain disorder characterized by declining language ability. It is a rare, often young-onset dementia with a devastating impact on the work and personal activities of those affected. At present there is no cure or disease-modifying therapy for PPA nor any way to arrest or slow the underlying progressive brain degeneration. Throughout the course of the condition any treatment must therefore be palliative-designed to manage symptoms and improve the quality of life of the affected person. The majority of those affected receive little or no follow-up care after diagnosis, particularly in the early stage of the disease. There is very little information in the medical literature about person-centered care designed to improve the quality of life of people with PPA written from the perspective of those living with this condition. I received an early and accurate clinical diagnosis of the nonfluent/agrammatic variant of PPA, supported by imaging. I am fortunate to have benefited from exemplary individualized care from a multidisciplinary medical team from the onset of my difficulties with language. In this paper, I discuss my lived experience of all aspects of this personalized and person-centered care, describing how it was founded on shared decision-making and a holistic, dementia-inclusive approach encompassing the physical, mental, emotional, psychosocial and spiritual dimensions of living with an incurable neurodegenerative disease., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Frontotemporal disorders are a group of rare young-onset dementias for which there is no cure, nor is there any way to slow the underlying progressive brain degeneration. To date those affected have typically received very little, if any, follow-up care after diagnosis, particularly in the early stages of their disease. I have received a clinical diagnosis, supported by imaging, of primary progressive aphasia, a form of frontotemporal degeneration characterized in the initial phase by progressive impairment of language ability. From the onset, I have been fortunate to receive excellent ongoing palliative care from a multidisciplinary team, some of whom had never previously seen anyone with this disorder. My quality of life has been enhanced by an evolving range of creative strategies and adaptations targeted to my deficits as they have arisen. In this paper, I discuss my experience of the process underlying this personalized plan, which serves as a paradigm for the development of novel palliative care approaches for people living with rare disorders, both neurodegenerative diseases and other conditions.

Background: Primary progressive aphasia (PPA) is a rare neurodegenerative brain disorder characterized by declining language ability. There is currently no way to reverse or slow the course of the progressive brain degeneration, nor is there a cure for PPA. Throughout the course of the disease, any treatment must therefore be palliative in nature and should be designed to manage symptoms and improve the quality of life of the affected person. There is little information in the medical literature about strategies to make meaningful improvements to the quality of life of people with PPA written from the perspective of those living with this condition., Aims: I have a clinical diagnosis of the nonfluent/agrammatic variant of PPA (nfvPPA), supported by imaging. In this report I discuss my experience of the progressive loss of language and communication skills, and detail the challenges I have been facing. I also describe how my quality of life has been enhanced by the early initiation of treatment focusing on communication strategies targeted to my specific impairments and designed to support my individual interests and goals., Methods & Procedures: I was fortunate to obtain an early diagnosis from a cognitive neurologist experienced with PPA. From the onset of my language difficulties, I have received excellent personalized care from a multidisciplinary medical team including speech-language pathologists, a cognitive neurologist and other doctors., Main Contributions: My life during the early stage of nfvPPA has been enriched by personalized care focused on supporting the particular activities, interests and goals that are most important and meaningful to me. As my disease has progressed, I have benefited from an evolving range of strategies and adaptations targeted to the specific deficits in the areas of speaking, writing and reading that I have been facing at any given time. In addition, I have adopted methods to enhance the benefit of these language-directed strategies. And I have been employing evidence-based approaches that improve general brain health and thereby indirectly support my language., Conclusions & Implications: My experience represents a model for the personalized care of people in the early stage of nfvPPA., What This Paper Adds: What is already known on the subject There is minimal information in the medical literature describing the subjective experience of a person with PPA. There is little information in the medical literature about strategies to make meaningful improvements to the quality of life of people in the early stage of PPA. What this paper adds to existing knowledge I have a clinical diagnosis of nfvPPA, supported by imaging. In this paper I give a first-person account of my experience of the progressive loss of language and communication skills, and I detail the challenges I have been facing. I describe how my quality of life during the early stage of nfvPPA has been enhanced by an evolving range of strategies and adaptations tailored to my speech and language deficits as they have arisen. These compensatory strategies have focused on supporting the particular activities, interests and goals that are most important and meaningful to me. What are the potential or actual clinical implications of this work? The description of my subjective experience of the progressive loss of language and communication skills offers insight for speech-language pathologists, neurologists and other professionals involved in the clinical care of people in the early stage of nfvPPA. My experience represents a model for the personalized clinical care of people in the early stage of this disorder., (© 2022 Royal College of Speech and Language Therapists.)

Primary progressive aphasia is a major clinical presentation of frontotemporal lobar degeneration and is a young-onset disorder characterized by deteriorating language skills. There is currently no cure for primary progressive aphasia, nor is it possible to slow the course of the underlying progressive brain degeneration. Hence the chief goal of treatment is palliative. Although the inability to employ language at one's previous level represents a significant functional impairment for those affected, there is a dearth of information about how to make meaningful improvements to the quality of life of people in the early stages of primary progressive aphasia. I have a clinical diagnosis, supported by imaging, of the nonfluent/agrammatic variant of primary progressive aphasia and am under the care of a multidisciplinary medical team. This report is based on my ongoing experience and describes the development and implementation of an evolving set of targeted strategies and adaptations designed to enhance the quality of life of a person in the early stages of this disorder.

Objective: The prevalence of bleeding disorders, notably von Willebrand disease (vWD), among adult women with objectively documented menorrhagia is consistently reported to be 10% to 20% and is even higher in adolescents presenting with menorrhagia., Options: Diagnostic tools and specific medical and, where appropriate, surgical alternatives to management are reviewed and evidence-based recommendations presented., Evidence: A MEDLINE search of the English literature between January 1975 and November 2003 was performed using the following key words: menorrhagia, uterine bleeding, pregnancy, von Willebrand, congenital bleeding disorder, desmopressin/DDAVP, tranexamic acid, oral contraceptives, medroxyprogesterone, therapy, hysterectomy, anesthesia, epidural, spinal. Recommendations from other society guidelines were reviewed., Values: The quality of evidence reported in this document has been described USing the Evaluation of Evidence criteria outlined in the Report of the Canadian Task Force on the Preventive Health Exam (Table 1). 13 RECOMMENDATIONS., (Copyright © 2018. Published by Elsevier Inc.)

Primary progressive aphasia (PPA) is a young-onset neurodegenerative disorder characterized by declining language ability. The nonfluent/agrammatic variant of PPA (PPA-G) has the core features of agrammatism in language production and effortful, halting speech. As with other frontotemporal spectrum disorders, there is currently no cure for PPA, nor is it possible to slow the course of progression. The primary goal of treatment is therefore palliative in nature. However, there is a paucity of published information about strategies to make meaningful improvements to the quality of life of people with PPA, particularly in the early stages of the disease where any benefit could most be appreciated by the affected person. This report describes a range of strategies and adaptations designed to improve the quality of life of a person with early-stage PPA-G, based on my experience under the care of a multidisciplinary medical team., (© The Author(s) 2014.)

Metastatic involvement of the skeleton is a frequent consequence of advanced prostate cancer. These skeletal metastases cause a number of debilitating complications and are refractory to current treatments. New therapeutic options are being explored, including conditionally replicating adenoviruses (CRAds). CRAds are engineered to selectively replicate in and destroy tumor cells and can be 'armed' with exogenous transgenes for enhanced potency. We hypothesized that a CRAd armed with osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, would inhibit the progression of prostate cancer bone metastases by directly lysing tumor cells and by reducing osteoclast activity. Although prostate cancer bone metastases are predominantly osteoblastic in nature, increased osteoclast activity is critical for the growth of these lesions. Ad5-Δ24-sOPG-Fc-RGD is a CRAd that carries a fusion of the ligand-binding domains of OPG and the Fc region of human IgG1 in place of the viral E3B genes. To circumvent low tumor cell expression of the native adenoviral receptor, an arginine-glycine-aspartic acid (RGD) peptide insertion within the viral fiber knob allows infection of cells expressing α(v) integrins. A 24-base pair deletion (Δ24) within viral E1A limits replication to cells with aberrant retinoblastoma cell cycle regulator/tumor suppressor expression. We have confirmed that Ad5-Δ24-sOPG-Fc-RGD replicates within and destroys prostate cancer cells and, in both murine and human coculture models, that infection of prostate cancer cells inhibits osteoclastogenesis in vitro. In a murine model, progression of advanced prostate cancer bone metastases was inhibited by treatment with Ad5-Δ24-sOPG-Fc-RGD but not by an unarmed control CRAd.

Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 10(10) v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15-40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression.

Purpose: Current treatments for ovarian cancer have limited therapeutic outcomes due to advanced stage of the disease at diagnosis. Among new therapies, conditionally replicating adenoviruses (CRAds), designed to selectively lyse cancer cells, hold promise. In clinical trials, CRAds exhibited limited efficacy thus far. Second-generation CRAds are being developed to express a therapeutic protein to enhance antitumor efficacy. One attractive target in the tumor microenvironment is the matrix metalloproteinases (MMPs) that degrade the extracellular matrix, and are upregulated in ovarian cancer. Tissue inhibitor of metalloproteinase 2 (TIMP2) is an endogenous inhibitor of MMPs. The present study developed and evaluated a novel CRAd (Ad5/3-CXCR4-TIMP2) for ovarian cancer therapy., Experimental Design: A targeted CRAd, Ad5/3-CXCR4-TIMP2 was developed using the CXCR4 promoter for enhanced replication, and expressing the TIMP2 transgene. The efficacy of this armed CRAd was determined in both established human ovarian cancer cell lines and in primary ovarian tumor samples., Results: Ad5/3-CXCR4-TIMP2 mediated expression of functional TIMP2, as demonstrated by the inhibition of MMP activity. In addition, arming with TIMP2 did not inhibit viral replication or oncolytic potency, as the TIMP2-armed viruses showed enhanced killing of cancer cells when compared to the unarmed viruses. We also examined viral replication in primary ovarian cancer tissues obtained from patients with stage III and IV ovarian cancer. In four of the five tumor samples, Ad5/3-CXCR4-TIMP2 revealed a 21- to 89-fold increase in replication when compared to the Ad5/3 virus., Conclusion: Results support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of patients with advanced ovarian cancer., (©2010 AACR.)

Osteoclasts are large, multinucleated cells responsible for the resorption of mineralized bone matrix. These cells are critical players in the bone turnover involved in bone homeostasis. Osteoclast activity is connected to the establishment and expansion of skeletal metastases from a number of primary neoplasms. Thus, the formation and activation of osteoclasts is an area of research with many potential avenues for clinical translation. Past studies of osteoclast biology have utilized primary murine cells cultured in vitro. Recently, techniques have been described that involve the generation of osteoclasts from human precursor cells. However, these protocols are often time-consuming and insufficient for generating large numbers of osteoclasts. We therefore developed a simplified protocol by which human osteoclasts may be easily and reliably generated in large numbers in vitro. In this study, osteoclasts were differentiated from bone marrow cells that had been aliquotted and frozen. Cells were generated by culture with recombinant macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Both human and murine RANKL were shown to efficiently generate osteoclasts, although higher concentrations of murine RANKL were required. Formation of osteoclasts was demonstrated qualitatively by tartrate-resistant acid phosphatase (TRAP) staining. These cells were fully functional, as confirmed by their ability to form resorption pits on cortical bone slices. Functional human osteoclasts can be difficult to generate in vitro by current protocols. We have demonstrated a simplified system for the generation of human osteoclasts in vitro that allows for large numbers of osteoclasts to be obtained from a single donor.

Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.

The coagulation system undergoes significant change during pregnancy. The clinician caring for the parturient must understand these changes, particularly when the parturient has a pre-existing haematological condition. Because many haematological conditions are rare, there often is limited information to guide the obstetric and anaesthetic management of these parturients., (2009 Elsevier Ltd. All rights reserved.)

Study Design: Ex vivo gene transfer for spinal fusion., Objective: This study aimed to evaluate ex vivo transfer of the nuclear-localized Hoxc-8-interacting domain of Smad1 (termed Smad1C) to rabbit bone marrow stromal cells (BMSCs) by a tropism-modified human adenovirus serotype 5 (Ad5) vector as a novel therapeutic approach for spinal fusion., Summary of Background Data: Novel approaches are needed to improve the success of bone union after spinal fusion. One such approach is the ex vivo transfer of a gene encoding an osteoinductive factor to BMSCs which are subsequently reimplanted into the host. We have previously shown that heterologous expression of the Hoxc-8-interacting domain of Smad1 in the nuclei of osteoblast precursor cells is able to stimulate the expression of genes related to osteoblast differentiation and induce osteogenesis in vivo. Gene delivery vehicles based on human Ad5 are well suited for gene transfer for spinal fusion because they can mediate high-level, short-term gene expression. However, Ad5-based vectors with native tropism poorly transduce BMSCs, necessitating the use of vectors with modified tropism to achieve efficient gene transfer., Methods: The gene encoding Smad1C was transferred to rabbit BMSCs by an Ad5 vector with native tropism or a vector retargeted to alphav integrins, which are abundantly expressed on rabbit BMSCs. Transduced BMSCs were maintained in osteoblastic differentiation medium for 30 days. Alkaline phosphatase activity was determined and cells stained for calcium deposition. As positive controls for osteogenesis, we used Ad5 vectors expressing bone morphogenetic protein 2. As negative controls, BMSCs were mock-transduced or transduced with an Ad5 vector expressing beta-galactosidase. In an immunocompetent rabbit model of spinal fusion, transduced BMSCs were coated onto absorbable gelatin sponge and implanted between decorticated transverse processes L6 and L7 of 8-week-old female New Zealand white rabbits. Animals were killed 4 weeks after implantation of the sponges, the fusion masses harvested and the area of new bone quantified using image analysis software., Results: The Smad1C-expressing tropism-modified Ad5 vector mediated a significantly higher level of alkaline phosphatase activity and calcium deposition in transduced rabbit BMSCs than all other vectors. The rabbit BMSCs transduced ex vivo with the Smad1C-expressing tropism-modified Ad5 vector mediated a greater amount of new bone formation than BMSCs transduced with any other vector., Conclusions: Delivery of the Smad1C gene construct to BMSCs by an alphav integrin-targeted Ad5 vector shows promise for spinal fusion and other applications requiring the formation of new bone in vivo.

We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

The purpose of the current study was to alter the broad native tropism of human adenovirus for virus targeting to c-erbB2-positive cancer cells. First, we engineered a single-chain antibody (scFv) against the c-erbB2 oncoprotein into minor capsid protein IX (pIX) of adenovirus serotype 5 (Ad5) in a manner commensurate with virion integrity and binding to the soluble extracellular c-erbB2 domain. To ablate native viral tropism and facilitate binding of the pIX-incorporated scFv to cellular c-erbB2, we replaced the Ad5 fiber with the Ad41 short (41s) fiber devoid of all known cell-binding determinants. The resultant Ad5F41sIX6.5 vector demonstrated increased cell binding and gene transfer as compared to the Ad5F41s control; however, this augmentation of virus infectivity was not c-erbB2 specific. Incorporation of a six-histidine (His(6)) peptide into the C-terminus of the 41s fiber protein resulted in markedly increased Ad5F41s6H infectivity in 293AR cells, which express a membrane-anchored scFv against the C-terminal oligohistidine tag, as compared to the Ad5F41s vector and the parental 293 cells. These data suggested that a 41s-fiber-incorporated His(6) tag could serve for attachment of an adapter protein designed to guide Ad5F41s6H infection in a c-erbB2-specific manner. We therefore engineered a bispecific scFv diabody (scDb) combining affinities for both c-erbB2 and the His(6) tag and showed its ability to provide up to 25-fold increase of Ad5F41s6H infectivity in c-erbB2-positive cells. Thus, Ad5 fiber replacement by a His(6)-tagged 41s fiber coupled with virus targeting mediated by an scDb adapter represents a promising strategy to confer Ad5 vector tropism for c-erbB2-positive cancer cells.

Malignant glioma, in particular glioblastoma multiforme (GBM), represents one of the most devastating cancers currently known and existing treatment regimens do little to change patient prognosis. Conditionally replicating adenoviral vectors (CRAds) represent attractive experimental anti-cancer agents with potential for clinical application. However, early protein products of the wild type adenovirus backbone--such as E1A--limit CRAds' replicative specificity. In this study, we evaluated the oncolytic potency and specificity of CRAds in which p300/CPB and/or pRb binding capacities of E1A were ablated to reduce non-specific replicative cytolysis. In vitro cytopathic assays, quantitative PCR analysis, Western blot, and flow cytometry studies demonstrate the superior anti-glioma efficacy of a double-mutated CRAd, Ad2/24CMV, which harbors mutations that reduce E1A binding to p300/CPB and pRb. When compared to its single-mutated and wild type counterparts, Ad2/24CMV demonstrated attenuated replication and cytotoxicity in representative normal human brain while displaying enhanced replicative cytotoxicity in malignant glioma. These results have implications for the development of double-mutated CRAd vectors for enhanced GBM therapy.

Hypotension is a common, treatable side effect of neuraxial anesthesia, which has significant side effects for the mother and demonstrable biochemical effects in the fetus. It is clear that a shift in management of hypotension in the obstetric population is in order, but we can only speculate on the benefits for the compromised fetus due to the lack of available information in that patient population.

Targeting gene expression to cancer cells remains a challenge for the development of gene and viral therapy for gliomas. Recent studies have highlighted transcriptional targeting as one of the possible solutions to overcome this limitation. In this context, melanoma associated antigens (MAAs) are usually over-expressed in brain tumors in comparison to normal brain tissue. For this reason, we investigated the use of the tyrosinase promoter as a transcriptional element to target oncolytic therapy for gliomas. Tyrosinase mRNA expression was evaluated by qRT-PCR in normal human brain tissue as well as in human glioma specimens. We found that this gene was significantly over-expressed in glioma cell lines and in primary glioma samples. Tyrosinase expression correlated with the grade of the tumor (p-value range: 0.05-0.001). Furthermore, transfection of several cell cultures with human and mouse tyrosinase promoters driving a luciferase reporter gene confirmed the activity of this promoter in mouse and human cells. To evaluate whether tyrosinase-activated conditionally replicative adenoviruses (CRAds) could induce toxicity in glioma cells, two vectors (Ad h/m and Ad24TYR) were tested in a mouse glioma model. C57BL/6 mice underwent intracranial injection of tumor cell line GL261. Survival was used to evaluate efficacy of the tested vectors. Mice receiving 1 x 10(9) MOI of Ad h/m and Ad24TYR following intracranial tumor implants had a median survival of 46+/-3 days (p<0.05); in contrast, those treated with medium had a median survival of 31+/-2 days. These results suggest that injection of tyrosinase CRAds leads to prolongation of survival in mice with experimental brain tumors. The tyrosinase promoter stands as a proof of principle of the potential use of MAA over-expression patterns for targeting novel anti-glioma therapies.

Purpose: Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo., Experimental Design: HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival., Results: Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone., Conclusion: Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Purpose: To review the effects of the long QT syndrome (LQTS) in the parturient and the current anesthetic management of patients with LQTS., Source: Relevant articles were obtained from a MEDLINE search spanning the years 1980-2006 and a PubMed search spanning the years 1949-2006. Bibliographies of retrieved articles were searched for additional articles., Principal Findings: The prevalence of LQTS in the developed world is one per 1,100 to 3,000 of the population. Clinically, LQTS is characterized by syncope, cardiac arrest and occasionally, by a history of seizures. The QT interval can also be prolonged by drugs, electrolyte imbalances, toxins and certain medical conditions. Long QT syndrome patients are at risk of torsades de pointes and ventricular fibrillation. Medical management aims to reduce dysrhythmia frequency. The LQTS is subdivided into different groups (LQT1-6) depending on the cardiac ion channel abnormality. Torsades can be precipitated by adrenergic stimuli such as stress or pain (LQT1 and 2), sudden noises (LQT2) or whilst sleeping (LQT3). Patients with LQTS require careful anesthetic management as they are at high risk of torsades perioperatively despite minimal data on the effects of anesthetic agents on the QT interval. While information on effects of LQTS in pregnancy is limited, the incidence of dysrhythmia increases postpartum. Isolated case reports of patients with LQTS women highlight several peripartum dysrhythmias., Conclusion: An understanding of LQTS and the associated risk factors contributing to dysrhythmias is important for anesthesthesiologists caring for parturients with LQTS.

Vectors based on human adenovirus serotypes 2 (Ad2) and 5 (Ad5) of species C possess a number of features that have favored their widespread employment for gene delivery both in vitro and in vivo. However, the use of recombinant Ad2- and Ad5-based vectors for gene therapy also suffers from a number of disadvantages. These vectors possess the tropism of the parental viruses, which infect all cells that possess the appropriate surface receptors, precluding the targeting of specific cell types. Conversely, some cell types that represent important targets for gene transfer express only low levels of the cellular receptors, which lead to inefficient infection. Another major disadvantage of Ad2- and Ad5-based vectors in vivo is the elicitation of both an innate and an acquired immune response. Considerable attention has therefore been focused on strategies to overcome these limitations, thereby permitting the full potential of adenoviral vectors to be realized.

Conditionally replicative adenoviruses (CRAds) represent novel therapeutic agents that have been recently applied in the context of breast cancer therapy. However, deficiencies in the ability of the adenovirus to infect target tumor cells and to specifically replicate within the tumor target represent key deficiencies preventing the realization of the full potential of this therapeutic approach. Minimal expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackie and adenovirus receptor) on breast cancer cells represents a major limitation for Ad5-based virotherapy. Genetic fiber chimerism is a method to alter the tropism of Ad5-based CRAds to achieve CAR-independent infectivity of tumor cells. Here, we describe the use of a CRAd with cancer specific transcriptional control of the essential Ad5 E1A gene using the human CXCR4 gene promoter. We further modified the fiber protein of this agent by switching the knob domain with that of the adenovirus serotype 3. The oncolytic activity of this 5/3 fiber-modified CRAd was studied in breast cancer cell lines, primary breast cancer and human liver tissue slices from patients, and in a xenograft breast cancer mouse model. This infectivity enhanced CRAd agent showed improved replication and killing in breast cancer cells in vitro and in vivo with a remarkable specificity profile that was strongly attenuated in nonbreast cancer cells, as well as in normal human breast and liver tissues. In conclusion, utilization of a CRAd that combined infectivity enhancement strategies and transcriptional targeting improved the CRAd-based antineoplastic effects for breast cancer therapy.

Clinical studies of replicating adenoviruses for the treatment of cancer have demonstrated their safety but have yielded disappointing results, indicating the need for new strategies to improve their efficacy. We hypothesized that the efficacy of a replicating adenovirus could be improved by expression of tissue inhibitor of metalloproteinases-2 (TIMP-2), a 21-kDa unglycosylated secretory protein. TIMP-2 specifically inhibits the active forms of a number of matrix metalloproteinases (MMPs) that play a role in the degradation of basement membranes and the extracellular matrix and are therefore involved in the control of the growth, invasion and metastasis of tumor cells, as well as angiogenesis. In addition, TIMP-2 can abrogate tumor growth and angiogenesis by a variety of mechanisms independent of MMP inhibition. In this study, we demonstrate that expression of TIMP-2 enhanced the antitumor efficacy of a replicating adenovirus in vivo, by reducing both tumor growth and angiogenesis.

Objective: The prevalence of bleeding disorders, notably von Willebrand disease (vWD), among adult women with objectively documented menorrhagia is consistently reported to be 10% to 20% and is even higher in adolescents presenting with menorrhagia. This consensus document has been developed by a multidisciplinary committee consisting of an anesthesiologist, 2 hematologists, and an obstetrician/gynaecologist and has been endorsed by their relevant specialty bodies. It has been prepared with the express purpose of providing guidelines for both women with inherited bleeding disorders and for their caregivers regarding the gynaecological and obstetric management of these women, including appropriate anesthesia support where indicated., Options: Diagnostic tools and specific medical and, where appropriate, surgical alternatives to management are reviewed and evidence-based recommendations presented., Evidence: A MEDLINE search of the English literature between January 1975 and November 2003 was performed using the following key words: menorrhagia, uterine bleeding, pregnancy, von Willebrand, congenital bleeding disorder, desmopressin/DDAVP, tranexamic acid, oral contraceptives, medroxyprogesterone, therapy, hysterectomy, anesthesia, epidural, spinal. Recommendations from other society guidelines were reviewed., Recommendations: 1. Inherited bleeding disorders should be considered in the differential diagnosis of all patients presenting with menorrhagia (II-2B). The graphical scoring system presented is a validated tool which offers a simple yet practical method that can be used by patients to quantify their blood loss (II-2B). 2. Because underlying bleeding disorders are frequent in women with menorrhagia, physicians should consider performing a hemoglobin/hematocrit, platelet count, ferritin, PT (INR) and APTT in women with menorrhagia. In women who have a personal history of other bleeding or a family history of bleeding, further investigation should be considered, including a vWD workup (factor VIII, vWF antigen, and vWF functional assay) (II-2B). 3. Treatment of menorrhagia in women with inherited bleeding disorders should be individualized (III-B). 4. An inherited bleeding disorder is not a contraindication to hormonal therapy (oral contraceptives [II-1B], depot medroxyprogesterone acetate (DMPA) [II-3B], danazol [II-2B], GnRH analogs [II-3B]) or local treatments (levonorgestrel-releasing IUS [II-1B]) and non-hormonal therapy (antifibrinolytic drug tranexamic acid [II-1B]) as well as desmopressin (II-1B). These therapies represent first line treatment. Blood products should not be used for women with mild bleeding disorders (III-A). 5. In women who no longer want to preserve their fertility, conservative surgical therapy (ablation) and hysterectomy may be options (III-B). Clinicians may consult the "SOGC Clinical Practice Guideline: Guidelines for the Management of Abnormal Uterine Bleeding" for an in-depth discussion of the available therapeutic modalities, both medical and surgical. To minimize the risk of intraoperative and post-operative hemorrhage, coagulation factors should be corrected preoperatively with post-operative monitoring (II-1B). 6. Girls growing up in families with a history of vWD or other inherited bleeding disorders should be tested pre-menarchally to determine whether or not they have inherited the disease to allow both the patient and her family to prepare for her first and subsequent menstrual periods (III-C). 7. In adolescents presenting with menorrhagia, an inherited bleeding disorder should be excluded (III-B). When possible, investigation should be undertaken before oral contraceptive therapy is instituted, as the hormonally induced increase in factor VIII and vWF may mask the diagnosis (II-B). 8. Pregnancy in women with inherited bleeding disorders may require a multidisciplinary approach. A copy of their recommendations should be given to the patient and she should be instructed to present it to the health care provider admitting her to the birthing centre. Women with severe bleeding disorders or with a fetus at risk for a severe bleeding disorder should deliver in a hospital (level three) or where there is access to consultants in obstetrics, anesthesiology, hematology, and pediatrics (III-C). 9. Vacuum extraction, forceps, fetal scalp electrodes, and fetal scalp blood sampling should be avoided if the fetus is known or thought to be at risk for a congenital bleeding disorder. A Caesarean section should be performed for obstetrical indications only (II-2C). 10. Epidural and spinal anesthesia are contraindicated if there is a coagulation defect. There is no contraindication to regional anesthesia if coagulation is normalized. The decision to use regional anesthesia should be made on an individual basis (III-C). 11. The risk of early and late postpartum hemorrhage is increased in women with bleeding disorders. Women with inherited bleeding disorders should be advised about the possibility of excessive postpartum bleeding and instructed to report this immediately (III-B). 12. Intramuscular injections, surgery, and circumcision should be avoided in neonates at risk for a severe hereditary bleeding disorder until adequate workup/preparation are possible (III-B). The quality of evidence reported in this document has been described using the Evaluation of Evidence criteria outlined in the Report of the Canadian Task Force on the Periodic Health Exam (Table 1).

Many clinically important tissues are refractory to adenovirus (Ad) infection due to negligible levels of the primary Ad5 receptor the coxsackie and adenovirus receptor CAR. Thus, development of novel CAR-independent Ad vectors should lead to therapeutic gain. Ovine atadenovirus type 7, the prototype member of genus Atadenovirus, efficiently transduces CAR-deficient human cells in vitro, and systemic administration of OAdV is not associated with liver sequestration in mice. The penton base of OAdV7 does not contain an RGD motif, implicating the long-shafted fiber molecule as a major structural dictate of OAdV tropism. We hypothesized that replacement of the Ad5 fiber with the OAdV7 fiber would result in an Ad5 vector with CAR-independent tropism in vitro and liver "detargeting" in vivo. An Ad5 vector displaying the OAdV7 fiber was constructed (Ad5Luc1-OvF) and displayed CAR-independent, enhanced transduction of CAR-deficient human cells. When administered systemically to C57BL/6 mice, Ad5Luc1-OvF reporter gene expression was reduced by 80% in the liver compared to Ad5 and exhibited 50-fold higher gene expression in the kidney than the control vector. To our knowledge, this is the first report of a fiber-pseudotyped Ad vector that simultaneously displays decreased liver uptake and a distinct organ tropism in vivo. This vector may have future utility in murine models of renal disease.

The utility of adenovirus serotype 5 (Ad5)-based vectors for gene therapy applications would be improved by cell-specific targeting. However, strategies to redirect Ad5 vectors to alternate cellular receptors via replacement of the capsid fiber protein have often resulted in structurally unstable vectors. In view of this, we hypothesized that the selection of modified adenoviruses during their rescue and propagation would be a straightforward approach that guarantees the generation of functional, targeted vectors. Based on our first generation fiber-fibritin molecule, several new chimeric fibers containing variable amounts of fibritin and the Ad5 fiber shaft were analyzed via a new scheme for Ad vector selection. Our selected chimera, composed of the entire Ad5 fiber shaft fused to the 12th coiled-coil segment of fibritin, is capable of efficient capsid incorporation and ligand display. Moreover, transduction by the resultant vector is independent of the expression of the native Ad5 receptor. The incorporation of the Fc-binding domain of Staphylococcus aureus protein A at the carboxy terminus of this chimeric fiber facilitates targeting of the vector to a variety of cellular receptors by means of coupling with monoclonal antibodies. In addition, we have concluded that Ad5 vectors incorporating individual targeting ligands require individual optimization of the fiber-fibritin chimera, which may be accomplished by selecting the optimal fiber-fibritin variant at the stage of rescue of the virus in cells of interest, as described herein.

Unlabelled: Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity., Introduction: Reduced BMD during spaceflight is partly caused by reduced bone formation. However, mechanisms responsible for this bone loss remain unclear. We have previously shown reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells (hMSCs) cultured in modeled microgravity (MMG). The small GTPase, RhoA, regulates actin stress fiber formation and has been implicated in the lineage commitment of hMSCs. We examined the effects of MMG on actin cytoskeletal organization and RhoA activity and the ability of constitutively active RhoA to reverse these effects., Materials and Methods: hMSCs were seeded onto plastic microcarrier beads at a density of 10(6) and allowed to form aggregates in DMEM containing 10% FBS for 7 days. Aggregates were incubated in DMEM containing 2% FBS for 6 h with or without an adenoviral vector containing constitutively active RhoA at a multiplicity of infection (moi) of 500 and allowed to recover in 10% FBS for 24 h. Cells were transferred to the rotary cell culture system to model microgravity or to be maintained at normal gravity for 7 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM beta-glycerol phosphate, and 50 muM ascorbic acid 2-phosphate., Results: F-actin stress fibers are disrupted in hMSCs within 3 h of initiation of MMG and are completely absent by 7 days, whereas monomeric G-actin is increased. Because of the association of G-actin with lipid droplets in fat cells, the observed 310% increase in intracellular lipid accumulation in hMSCs cultured in MMG was not unexpected. Consistent with these changes in cellular morphology, 7 days of MMG significantly reduces RhoA activity and subsequent phosphorylation of cofilin by 88+/-2% and 77+/-9%, respectively. Importantly, introduction of an adenoviral construct expressing constitutively active RhoA reverses the elimination of stress fibers, significantly increases osteoblastic gene expression of type I collagen, alkaline phosphatase, and runt-related transcription factor 2, and suppresses adipocytic gene expression of leptin and glucose transporter 4 in hMSCs cultured in MMG., Conclusion: Suppression of RhoA activity during MMG represents a novel mechanism for reduced osteoblastogenesis and enhanced adipogenesis of hMSCs.

Objective: The prevalence of bleeding disorders, notably von Willebrand disease (vWD), among adult women with objectively documented menorrhagia is consistently reported to be 10% to 20% and is even higher in adolescents presenting with menorrhagia. This consensus document has been developed by a multidisciplinary committee consisting of an anesthesiologist, 2 hematologists, and an obstetrician/gynaecologist and has been endorsed by their relevant specialty bodies. It has been prepared with the express purpose of providing guidelines for both women with inherited bleeding disorders and for their caregivers regarding the gynaecological and obstetric management of these women, including appropriate anesthesia support where indicated., Options: Diagnostic tools and specific medical and, where appropriate, surgical alternatives to management are reviewed and evidence-based recommendations presented., Evidence: A MEDLINE search of the English literature between January 1975 and November 2003 was performed using the following key words: menorrhagia, uterine bleeding, pregnancy, von Willebrand, congenital bleeding disorder, desmopressin/DDAVP, tranexamic acid, oral contraceptives, medroxyprogesterone, therapy, hysterectomy, anesthesia, epidural, spinal. Recommendations from other society guidelines were reviewed., Recommendations: 1. Inherited bleeding disorders should be considered in the differential diagnosis of all patients presenting with menorrhagia (II-2B). The graphical scoring system presented is a validated tool which offers a simple yet practical method that can be used by patients to quantify their blood loss (II-2B). 2. Because underlying bleeding disorders are frequent in women with menorrhagia, physicians should consider performing a hemoglobin/hematocrit, platelet count, ferritin, PT (INR) and APTT in women with menorrhagia. In women who have a personal history of other bleeding or a family history of bleeding, further investigation should be considered, including a vWD workup (factor VIII, vWF antigen, and vWF functional assay) (II-2B). 3. Treatment of menorrhagia in women with inherited bleeding disorders should be individualized (III-B). 4. An inherited bleeding disorder is not a contraindication to hormonal therapy (oral contraceptives [II-1B], depot medroxyprogesterone acetate (DMPA) [II-3B], danazol [II-2B], GnRH analogs [II-3B]) or local treatments (levonorgestrel-releasing IUS [II-1B]) and non-hormonal therapy (antifibrinolytic drug tranexamic acid [II-1B]) as well as desmopressin (II-1B). These therapies represent first line treatment. Blood products should not be used for women with mild bleeding disorders (III-A). 5. In women who no longer want to preserve their fertility, conservative surgical therapy (ablation) and hysterectomy may be options (III-B). Clinicians may consult the "SOGC Clinical Practice Guideline: Guidelines for the Management of Abnormal Uterine Bleeding" for an in-depth discussion of the available therapeutic modalities, both medical and surgical. To minimize the risk of intraoperative and post-operative hemorrhage, coagulation factors should be corrected preoperatively with post-operative monitoring (II-1B). 6. Girls growing up in families with a history of vWD or other inherited bleeding disorders should be tested pre-menarchally to determine whether or not they have inherited the disease to allow both the patient and her family to prepare for her first and subsequent menstrual periods (III-C). 7. In adolescents presenting with menorrhagia, an inherited bleeding disorder should be excluded (III-B). When possible, investigation should be undertaken before oral contraceptive therapy is instituted, as the hormonally induced increase in factor VIII and vWF may mask the diagnosis (II-B). 8. Pregnancy in women with inherited bleeding disorders may require a multidisciplinary approach. A copy of their recommendations should be given to the patient and she should be instructed to present it to the health care provider admitting her to the birthing centre. Women with severe bleeding disorders or with a fetus at risk for a severe bleeding disorder should deliver in a hospital (level three) or where there is access to consultants in obstetrics, anesthesiology, hematology, and pediatrics (III-C). 9. Vacuum extraction, forceps, fetal scalp electrodes, and fetal scalp blood sampling should be avoided if the fetus is known or thought to be at risk for a congenital bleeding disorder. A Caesarean section should be performed for obstetrical indications only (II-2C). 10. Epidural and spinal anesthesia are contraindicated if there is a coagulation defect. There is no contraindication to regional anesthesia if coagulation is normalized. The decision to use regional anesthesia should be made on an individual basis (III-C). 11. The risk of early and late postpartum hemorrhage is increased in women with bleeding disorders. Women with inherited bleeding disorders should be advised about the possibility of excessive postpartum bleeding and instructed to report this immediately (III-B). 12. Intramuscular injections, surgery, and circumcision should be avoided in neonates at risk for a severe hereditary bleeding disorder until adequate workup/preparation are possible (III-B). The quality of evidence reported in this document has been described using the Evaluation of Evidence criteria outlined in the Report of the Canadian Task Force on the Periodic Health Exam (Table 1).

The use of adenovirus type 5 (Ad5) for cancer therapy is limited by deficiency of its primary cell attachment receptor, coxsackie and adenovirus receptor (CAR), on cancer cells. Ad5 retargeting to alternate receptors through fiber genetic modification can be used to circumvent CAR dependence of its tropism, and thereby achieve infectivity enhancement. Here we propose and test a novel "complex mosaicism" approach for fiber modification, which combines serotype chimerism with peptide ligand(s) incorporation in a single-fiber molecule. We incorporated integrin-binding peptide RGD-4C in the HI-loop, at the carboxy (C)-terminus, or both locales of the Ad3 knob, in the context of Ad5/3 chimera fiber in order to retarget simultaneously the Ad vector to integrins and Ad3 receptors. The infectivity enhancement of the fiber modifications was assessed in various cancer cell lines as cancer-targeting models. Replication-defective complex mosaic Ad-luc vectors bearing chimeric fiber (F.5/3), with or without C-terminal RGD-modification of Ad3 knob, demonstrated up to 55-fold gene transfer increase in bladder cancer cell lines. Although this augmentation was primarily due to Ad3 receptor targeting, some contribution of RGD-mediated integrin-targeting was also observed, suggesting that complex mosaic modification can function in a dual-receptor targeting via a single Ad3 fiber knob.

Dendritic cells (DCs) are a central element in the development of antigen-specific immune responses. The lack of a specific and efficient technique for the in vivo delivery of antigens to DCs remains a major obstacle limiting a vaccine's ability to induce an effective immune response. The efficacy of adenoviral (Ad) vectors in this regard can be enhanced through alterations in vector tropism such that DC-targeted transduction is achieved. Here, the efficiency of DC transduction by Ad vectors retargeted to DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) was studied and compared to that of Ad vectors retargeted through CD40. A comparable and significant enhancement of gene transfer to monocyte derived DCs (MDDCs) was accomplished by means of an Ad vector harboring the Fc-binding domain of Staphylococcus aureus protein A in combination with antibodies to DC-SIGN or to CD40 or with fused complexes of human Ig-Fc with their natural ligands, i.e., ICAM-3 or CD40L, respectively. Whereas CD40-targeted Ad transduction resulted in a more profound phenotypic DC maturation, DC-SIGN- and CD40-targeted Ad both induced similar levels of IL-12 secretion. These data demonstrate the usefulness of DC-SIGN as a DC-restricted targeting motif for Ad-mediated vaccination strategies.

Purpose: To report the first use of spinal anesthesia for Cesarean section (CS) in a parturient with a long QT syndrome (LQTS) and an automatic implantable cardiac defibrillator (AICD). Although both general and epidural anesthesia have been described for CS in patients with LQTS, there are no previous case reports on the use of spinal anesthesia. The clinical features, diagnosis, treatment and anesthetic management of LQTS are discussed., Clinical Features: A 31-yr-old woman, gravida 2 para 1 known to have LQTS and an AICD, presented in labour at 35 weeks gestation, three weeks before her scheduled CS. Her previous delivery by CS under spinal anesthesia at our institution was uneventful. On this occasion, we elected to administer spinal anesthesia because she was asymptomatic (no arrhythmia or cardiac arrest) for the last few years, was hemodynamically stable, and had received uneventful spinal anesthesia before., Conclusion: Spinal anesthesia was used safely for CS in this parturient with LQTS.

Many clinically relevant tissues are refractory to Ad5 transduction because of negligible levels of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR). Thus, development of Ad vectors that display CAR-independent tropism could lead directly to therapeutic gain. The Toronto strain of canine adenovirus type 2 (CAV2) exhibits native tropism that is augmented by, but not fully dependent upon, CAR for cellular transduction. We hypothesized that an Ad5 vector containing the nonhuman CAV2 knob would provide expanded tropism and constructed Ad5Luc1-CK, an E1-deleted Ad5 vector encoding the fiber knob domain from CAV2. Ad5Luc1-CK gene delivery to CAR-deficient cells was augmented up to 30-fold versus the Ad5 control vector, and correlated with increased cell surface binding. Further, we confirmed the importance of cellular integrins to Ad5Luc1-CK transduction. Herein, we present the rationale, design, purification, and characterization of a novel tropism modified, infectivity-enhanced Ad vector.

Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their efficacy and safety profile. Liver tropism is important with regard to potential clinical toxicity in humans. Ad5/3 chimeras in which the Ad5 knob is substituted by the Ad3 knob, such as Ad5/3luc1, have been recently shown to increase infectivity of ovarian cancer cell lines and primary tumor cells, which express low levels of the coxsackie-adenovirus receptor (CAR), without increasing infectivity of liver cells. A novel strategy to address the problem of liver uptake and improve the tumor/liver ratio is genetic replacement of the Ad fiber shaft. Ad5.Ad3.SH.luc1 is an Ad5-based vector that contains the fiber shaft from Ad serotype 3 but the fiber knob from Ad serotype 5. To compare tumor/liver of Ad5.Ad3.SH.luc1 and Ad5/3luc1 in vivo, we created three different tumor and treatment models of ovarian cancer in mice, simulating intraperitoneal and intravenous administration of tumors. Ad5.Ad3.SH.luc1 displayed the lowest liver tropism of all viruses in all models tested. Intravenous administration of all viruses resulted in higher tumor transduction rates compared to intraperitoneal administration. Genetic shortening of the Ad5 fiber shaft significantly increases relative tumor/liver gene transfer. This could improve the effective tumor dose and reduce side effects, thereby increasing the bioavailability of therapeutic agents.

Gene therapy has rapidly evolved into a field that is treating not only inborn errors of metabolism, but other diseases associated with poor outcomes such as malignancy, where transient gene expression can be therapeutic. Cancer gene therapy is a novel form of treatment that exploits differences at the molecular level between normal and malignant cells. Current gene therapy approaches that are being evaluated include the use of replication competent viruses, mutation compensation strategies, improved targeting with tumor specific promoters, and the utilization of enhanced infectivity viruses. An additional aspect of gene therapy that has gained increased interest in the last several years is the utilization of single-chain antibodies (scFv). Specifically, scFv have been utilized to target molecular processes associated with carcinogenesis, as well as to improve gene transfer efficiency. We will limit our discussion to the role of scFv in targeting molecular processes associated with carcinogenesis.

Adenoviral vectors can be employed for gene delivery to skeletal muscle, both ex vivo and in vivo. Although the realization of the full potential of adenoviral vectors awaits the development of methods to allow safe and efficient targeted gene delivery to mature skeletal muscle upon intravenous vector administration (1), the current generation of vectors has nonetheless found utility in preclinical studies of gene therapy and in gene-transfer experiments designed to study muscle biology. Features of adenoviral vectors that have favored their use for gene delivery to skeletal muscle include the ability to infect both actively dividing and terminally differentiated cells, as well as their large insert capacity. Gutted adenoviral vectors are capable of carrying the large dystrophin gene together with regulatory sequences, and are therefore appropriate vehicles for gene-replacement therapy for Duchenne muscular dystrophy. In addition to their suitability for in vivo gene-therapy applications, adenoviral vectors have been used ex vivo to transfer genes to myoblasts prior to myoblast transplantation into muscle.

Dendritic cells (DCs) are "professional" antigen-presenting cells (APCs) that are uniquely capable of activating and instructing a naive immune system to mount a specific cellular and humoral response. Recognition of this crucial function makes the development of technologies for DC-based immuno-therapies a priority for the treatment of a wide variety of diseases. The most immediate impact of this emerging technology will be in the treatment of cancer and the development of third generation vaccines to protect against viral and intracellular pathogens. In addition to elicitation of immune responses, DCs also function to maintain tolerance to "self." Once the biological basis for this important function is understood, future applications of DC-based immuotherapies may be developed to ameliorate autoimmune diseases or enhance acceptance of transplanted organs. The feasibility of "engineering" the function of DCs has been realized by recent advances in ex vivo methodologies that allow selective DC propagation, antigen loading, and genetic modification in vitro for subsequent therapeutic transfer into the host. Ultimately, the ability to genetically modify these cells will allow us to design DC-mediated interventions that will direct predictable control of either immune activation or tolerance in vivo.

Adenoviruses, which were first isolated in the 1950s, have been developed as gene-delivery vehicles, or vectors, since the early 1980s. The adenoviruses constitute the Adenoviridae family, which is divided into two genera: the Aviadenovirus genus infects only birds, whereas the Mastadenovirus genus contains viruses that infect a range of mammalian species. Human adenoviruses are classified into six subgroups based on the percentage of guanine and cytosine in the DNA molecules and the ability to agglutinate red blood cells. They are further subdivided into more than 50 serotypes, primarily on the basis of neutralization assays.

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA-mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild-type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma., (Copyright 2004 Wiley-Liss, Inc.)

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.

The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction. Furthermore, an Ad5-based chimeric vector, designated Ad5/3, containing a chimeric fiber proteins possessing the Ad3 knob, demonstrates CAR-independent tropism by virtue of targeting the Ad3 receptor. Based on these findings, we hypothesized that a mosaic virus possessing both the Ad5 knob and the Ad3 knob on the same virion could utilize either primary receptor, resulting in expanded tropism. In this study, we generated a dual-knob mosaic virus by coinfection of 293 cells with Ad5-based and Ad5/3-based vectors. Characterization of the resultant virions confirmed the incorporation of both Ad5 and Ad3 knobs in the same particle. Furthermore, this mosaic virus was able to utilize either receptor, CAR and the Ad3 receptor, for virus attachment to cells. Enhanced Ad infectivity with the mosaic virus was shown in a panel of cell lines, with receptor profiles ranging from CAR-dominant to Ad3 receptor-dominant. Thus, this mosaic virus strategy may offer the potential to improve Ad-based gene therapy approaches by infectivity enhancement and tropism expansion.