Plant toxins are sequestered by many animals and the toxicity is frequently advertised by aposematic displays to deter potential predators. Such ‘unpalatability by appropriation’ is common in many invertebrate groups and also found in a few vertebrate groups. However, potentially lethal toxicity by acquisition has so far never been reported for a placental mammal. Here, we describe complex morphological structures and behaviours whereby the African crested rat, Lophiomys imhausi, acquires, dispenses and advertises deterrent toxin. Roots and bark of Acokanthera schimperi (Apocynaceae) trees are gnawed, masticated and slavered onto highly specialized hairs that wick up the compound, to be delivered whenever the animal is bitten or mouthed by a predator. The poison is a cardenolide, closely resembling ouabain, one of the active components in a traditional African arrow poison long celebrated for its power to kill elephants.

Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6–8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.

Introduction

A number of questions remain unanswered in the field of cell therapy for acute myocardial infarction, including what is the optimal cell type, and can therapeutic efficacy be enhanced by conditioning regimens. In this study, we sought to address these questions by directly comparing the effect of bone marrow-derived mesenchymal stem cells and unrestricted somatic stem cells delivered 24 hours post-myocardial infarction and by determining if the therapeutic efficacy of unrestricted somatic stem cells could be enhanced by exposing the cells to guiding factors before cell transplantation.

Methods

Unrestricted somatic stem cells were guided by exposure to 50 ng/mL basic fibroblast growth factor, 20 ng/mL hepatocyte growth factor and 20 ng/mL bone morphogenetic protein-2 for 24 hours. Using a Sprague-Dawley rat model of acute myocardial infarction, we transplanted cells by intramyocardial injection 24 hours post-myocardial infarction. Cardiac function was serially measured using echocardiography, and histological analyses of infarct morphology, angiogenesis and apoptosis were obtained. Transcriptomic and proteomic changes were assessed using microarray and real-time quantitative PCR.

Results

When assessed 28 days after the myocardial infarction, the delivery of mesenchymal stem cells 24 hours post-myocardial infarction did not improve ejection fraction (P = 0.19), and did not prevent the decline in ejection fraction observed in the absence of cell therapy (P = 0.17). The administration of unrestricted somatic stem cells also did not improve ejection fraction (P = 0.11), but did prevent a further decline in ejection fraction (P = 0.001). Delivery of guided unrestricted somatic stem cells significantly improved ejection fraction (P = 0.03). Guided unrestricted somatic stem cells restored function to a greater extent than mesenchymal stem cells (P = 0.03). The infarct area (P = 0.2), apoptosis (P = 0.07) and angiogenesis (P = 0.09) did not differ between groups. Microarray analysis revealed that, following pre-implantation guiding, the gene groupings of mitosis, signalling and angiogenesis were highly overrepresented, mediators of apoptosis were overrepresented, and cardiomyocyte-associated genes were not differentially expressed.

Conclusions

These results suggest that guided unrestricted somatic stem cells have a moderate capacity to repair cardiac damage and that they are more effective than mesenchymal stem cells in restoring cardiac function after a myocardial infarction. The mechanism of the benefit was not fully elucidated in this study, but these observations may be mediated by favorable dysregulation of angiogenic and apoptotic gene groupings.

Hepatocyte-like cells derived from stem cells hold great potential for clinical and pharmaceutical applications, including high-throughput drug toxicity screening. We report a three-dimensional aggregate culture system for the directed differentiation of adult rat bone marrow-derived stem cells, rat multipotent adult progenitor cells, to hepatocyte-like cells. Compared to adherent monolayer cultures, differentiation in the aggregate culture system resulted in significantly higher expression level of liver-specific transcripts, including an increased albumin mRNA level, and higher levels of albumin and urea secretion. This coincides with the presence of significantly more cells that express intracellular albumin at levels found in primary hepatocytes. The differentiated cell aggregates exhibited cytochrome P450-mediated ethoxyresorufin-O-dealkylation and pentoxyresorufin-O-dealkylation activity. Consistent with these increased mature functions, cells within the aggregates were shown to have many ultrastructural features of mature hepatocytes by transmission electron microscopy. With the scalability of the aggregate culture system and the enhanced differentiation capability, this system may facilitate translation of generating hepatocytes from stem cells to technology.

Adult mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential which makes them attractive targets for regenerative medicine applications. However, to date, therapeutic success of MSC-therapy is limited and the genetic modification of MSCs using viral vectors is one option to improve their therapeutic potential. Ex-vivo genetic modification of MSCs using recombinant adenovirus (Ad) could be promising to reduce undesired immune responses as Ad will be removed before cell/tissue transplantation. In this regard, we investigated whether Ad-modification of MSCs alters their immunological properties in vitro and in vivo. We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86. Moreover, Ad-transduction caused no significant changes in terms of pro-inflammatory cytokine expression, chemokine and chemokine receptor and Toll-like receptor expression. In addition, Ad-modification of MSCs had no affect on their ability to suppress T cell proliferation in vitro. In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues. Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.

Background

In order to facilitate multinational clinical research, regulatory requirements need to become international and harmonised. The EU introduced the Directive 2001/20/EC in 2004, regulating investigational medicinal products in Europe.

Methods

We conducted a survey in order to identify the national regulatory requirements for major categories of clinical research in ten European Clinical Research Infrastructures Network (ECRIN) countries-Austria, Denmark, France, Germany, Hungary, Ireland, Italy, Spain, Sweden, and United Kingdom-covering approximately 70% of the EU population. Here we describe the results for regulatory requirements for typical investigational medicinal products, in the ten countries.

Results

Our results show that the ten countries have fairly harmonised definitions of typical investigational medicinal products. Clinical trials assessing typical investigational medicinal products require authorisation from a national competent authority in each of the countries surveyed. The opinion of the competent authorities is communicated to the trial sponsor within the same timelines, i.e., no more than 60 days, in all ten countries. The authority to which the application has to be sent to in the different countries is not fully harmonised.

Conclusion

The Directive 2001/20/EC defined the term 'investigational medicinal product' and all regulatory requirements described therein are applicable to investigational medicinal products. Our survey showed, however, that those requirements had been adopted in ten European countries, not for investigational medicinal products overall, but rather a narrower category which we term 'typical' investigational medicinal products. The result is partial EU harmonisation of requirements and a relatively navigable landscape for the sponsor regarding typical investigational medicinal products.

Background

Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes.

Objective

The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present.

Methods

Single-cell suspensions of neoplastic cells were prepared from biopsy samples and fine-needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser-excitable (405 nm) membrane-permeable DNA-binding fluorescent dye (DyeCycle Violet, DCV), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7-amino-actinomycin D, an argon-excitable (488 nm) membrane-impermeable DNA-binding fluorescent dye. Multi-parameter flow cytometry was used for analysis based on selective uptake and laser-activated fluorescence of these dyes.

Results

Cytoplasmic fragments, which were DCV-negative and CD45-positive, and dead cells, which were positive for 7-amino-actinomycin D, were efficiently separated from neoplastic cells.

Conclusion

Staining with DyeCycle Violet is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma.

While stem cell transplantation could potentially treat a variety of disorders, clinical studies have not yet demonstrated conclusive benefits. This may be partly because transplanted stem cells have low survival rates, potentially due to host inflammation. The system described herein used two different gene therapy techniques to improve retention of rat mesenchymal stem cells. In the first, stem cells were transfected with interleukin-10 before being loaded into a collagen scaffold. In the second, unmodified stem cells were loaded into a collagen scaffold along with polymer-complexed interleukin-10 plasmids. The scaffolds were surgically implanted into the dorsum of syngeneic rats. At each endpoint, the scaffolds were explanted and cell retention, interleukin-10 level and inflammatory response were quantified. All treatment groups had statistically significant increases in cell retention after 7 days, but the group treated with 2 μg of interleukin-10 polyplexes had a significant improvement even at 21 days. This cell retention was associated with increased interleukin-10 and decreased levels of pro-inflammatory cytokines and apoptosis. The primary effect on the inflammatory response appeared to be on macrophage differentiation, encouraging the regulatory phenotype over the cytotoxic lineage. Improving cell survival may be an important step towards realization of the therapeutic potential of stem cells.

Mesenchymal stem cells have a natural tropism for tumours and their metastases, and are also considered immunoprivileged. This remarkable combination of properties has formed the basis for many studies investigating their potential as tumour-specific delivery vehicles for suicide genes, oncolytic viruses and secreted therapeutic proteins. The aim of the present review is to discuss the range of approaches that have been used to exploit the tumour-homing capacity of mesenchymal stem cells for gene delivery, and to highlight advances required to realize the full potential of this promising approach.

A 40-year-old woman presented with a 10 day history of episodic vagueness, speech disturbance and blurred vision. Episodes typically occurred in the morning after awaking from sleep and resolved with food ingestion. She had no past medical history, did not drink alcohol and was not on any medication. Physical examination was normal with no evidence of endocrinopathy. After 10 h of fasting, she became hypoglycaemic with evidence of neuroglycopenia, which resolved with intravenous dextrose. Biochemical investigations revealed decreased glucose, insulin and C-peptide values with an increased excess insulin-like growth factor II: excess insulin-like growth factor I (IGF-II: IGF-I) ratio. Radiological examinations of the abdomen and pelvis revealed a heterogenous 10.5 cm left renal mass. The patient underwent a radical left nephrectomy. She had complete resolution of hypoglycaemic events. Histology revealed a renal sarcoma, grade 2/3. This is the first report in the literature involving a renal sarcoma causing non-islet cell tumour hypoglycaemia via excess IGF-II secretion.

We have previously shown that immunotherapy directed against the protein Nogo-A leads to recovery on a skilled forelimb reaching task in rats after sensorimotor cortex stroke, which correlated with axonal and dendritic plasticity. Here we investigated anti-Nogo-A immunotherapy as an intervention to improve performance on a spatial memory task in aged rats after stroke, and whether cognitive recovery was correlated with structural plasticity. Aged rats underwent a unilateral distal permanent middle cerebral artery occlusion and one week later were treated with an anti-Nogo-A or control antibody. Nine weeks post-stroke, treated rats and normal aged rats were tested on the Morris water maze task. Following testing rats were sacrificed and brains processed for the Golgi-Cox method. Hippocampal CA3 and CA1 pyramidal and dentate gyrus granule cells were examined for dendritic length and number of branch segments, and CA3 and CA1 pyramidal cells were examined for spine density and morphology. Anti-Nogo-A immunotherapy given one week following stroke in aged rats improved performance on the reference memory portion of the Morris water maze task. However, this improved performance was not correlated with structural changes in the hippocampal neurons examined. Our finding of improved performance on the Morris water maze in aged rats after stroke and treatment with anti-Nogo-A immunotherapy demonstrates the promising therapeutic potential for anti-Nogo-A immunotherapy to treat cognitive deficits after stroke. The identification of sites of axonal and dendritic plasticity in the aged brain after stroke and treatment with anti-Nogo-A immunotherapy is still under investigation.

Introduction

A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.

Methods

Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.

Results

The second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.

Conclusions

Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

Evolution of facial morphology arises from variation in the activity of developmental regulatory networks that guide the formation of specific craniofacial elements. Importantly, the acquisition of novel morphology must be integrated with a phylogenetically inherited developmental program. We have identified a unique region of the secondary palate associated with the periodic formation of rugae during the rostral outgrowth of the face. Rugae function as SHH signaling centers to pattern the elongating palatal shelves. We have found that a network of signaling genes and transcription factors is spatially organized relative to palatal rugae. Additionally, the first formed ruga is strategically positioned at the presumptive junction of the future hard and soft palate that defines anterior-posterior differences in regional growth, mesenchymal gene expression and cell fate. We propose a molecular circuit integrating FGF and BMP signaling to control proliferation and differentiation during the sequential formation of rugae and inter-rugae domains in the palatal epithelium. The loss of p63 and Sostdc1 expression and failed rugae differentiation highlight that coordinated epithelial mesenchymal signaling is lost in the Fgf10 mutant palate. Our results establish a genetic program that reiteratively organizes signaling domains to coordinate the growth of the secondary palate with the elongating midfacial complex.

This review highlights current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury. The concept of developing 3-dimensional polymer scaffolds for placement into a spinal cord transection model has recently been more extensively explored as a solution for restoring neurologic function after injury. Given the patient morbidity associated with respiratory compromise, the discrete tracts in the spinal cord conveying innervation for breathing represent an important and achievable therapeutic target. The aim is to derive new neuronal tissue from the surrounding, healthy cord that will be guided by the polymer implant through the injured area to make functional reconnections. A variety of naturally derived and synthetic biomaterial polymers have been developed for placement in the injured spinal cord. Axonal growth is supported by inherent properties of the selected polymer, the architecture of the scaffold, permissive microstructures such as pores, grooves or polymer fibres, and surface modifications to provide improved adherence and growth directionality. Structural support of axonal regeneration is combined with integrated polymeric and cellular delivery systems for therapeutic drugs and for neurotrophic molecules to regionalize growth of specific nerve populations.

Background

'Compassionate use' programmes allow medicinal products that are not authorised, but are in the development process, to be made available to patients with a severe disease who have no other satisfactory treatment available to them. We sought to understand how such programmes are regulated in ten European Union countries.

Methods

The European Clinical Research Infrastructures Network (ECRIN) conducted a comprehensive survey on clinical research regulatory requirements, including questions on regulations of 'compassionate use' programmes. Ten European countries, covering approximately 70% of the EU population, were included in the survey (Austria, Denmark, France, Germany, Hungary, Ireland, Italy, Spain, Sweden, and the UK).

Results

European Regulation 726/2004/EC is clear on the intentions of 'compassionate use' programmes and aimed to harmonise them in the European Union. The survey reveals that different countries have adopted different requirements and that 'compassionate use' is not interpreted in the same way across Europe. Four of the ten countries surveyed have no formal regulatory system for the programmes. We discuss the need for 'compassionate use' programmes and their regulation where protection of patients is paramount.

Conclusions

'Compassionate use' is a misleading term and should be replaced with 'expanded access'. There is a need for expanded access programmes in order to serve the interests of seriously ill patients who have no other treatment options. To protect these patients, European legislation needs to be more explicit and informative with regard to the regulatory requirements, restrictions, and responsibilities in expanded access programmes.

In a chemical mutagenesis screen we identified Szt2 (seizure threshold 2) as a gene that confers low seizure threshold to mice and may also enhance epileptogenesis. The semidominant phenotype was mapped to Chromosome 4 and narrowed further to a critical interval of approximately 650 kb. A novel large (>10 kb) transcript in the critical interval was found to have four-fold increased steady-state expression at the RNA level in Szt2 homozygous mutant brain. The corresponding 72 exon gene encodes a 378 kD protein with no significant or suggestive sequence similarities to any other protein. The mutant allele of Szt2 contains a splice donor mutation after exon 32, predicting transcriptional read-through, translational frameshift and premature stop. A second Szt2 allele, containing a gene-trap mutation in exon 21, also conferred a low seizure threshold and increased RNA expression, but unlike the ENU allele, some gene-trap homozygotes died embryonically. Szt2 is transcribed in many tissues, with the highest expression in brain, and it is also expressed during embryonic development. Szt2 is highly conserved in evolution, with a clear, single orthologue found in all land vertebrates and in many invertebrates. Interestingly, in mammals the Szt2 gene resides in a highly conserved head-to-head configuration with Med8 (which encodes a Mediator complex subunit), separated by only 91 nt. While the biological function of Szt2 remains unknown, its high conservation, unique structure and effect on seizure threshold suggest that it serves an important role in the central nervous system.

Background

Ovarian cancer is frequently diagnosed at an advanced stage, and although initially responsive to surgery and chemotherapy, has a high rate of recurrence and mortality. Cellular immunotherapy may offer the prospect of treatment to prevent or delay recurrent metastatic disease.

Objective

To provide an overview of current innovations in cellular immunotherapy for ovarian cancer, with an emphasis on dendritic cell vaccination and adoptive T cell immunotherapy.

Methods

Three key areas are explored in this review. First, an appraisal of the current state of the art of cellular immunotherapy for treatment of ovarian cancer. Second, a discussion of the immunological defenses erected by ovarian cancer to prevent immunological attack, with an emphasis on the role of tumor-associated regulatory T cells. Third, an exploration of innovative techniques that may enhance the ability of cellular immunotherapy to overcome ovarian tumor-associated immune suppression.

Results/conclusion

Ovarian cancer is recognized as a paradigm for tumor-associated immune suppression. Innovative approaches for antagonism of tumor-associated regulatory T cell infiltration and redirection of self antigen-driven regulatory T cell activation may provide the key to development of future strategies for cellular immunotherapy against ovarian cancer.

Deficits in learning, memory, and executive functions are common cognitive sequelae of Parkinson's disease with dementia (PDD) and Alzheimer's disease (AD); however, the pattern of deficits within these populations is distinct. Hierarchical regression was used to investigate the contribution of two measures with executive function properties (Verbal Fluency and CLOX) on list-learning performance (CVLT-II total words learned) in a sample of 25 PDD patients and 25 matched AD patients. Executive measures were predictive of list learning in the PDD group after the contribution of overall cognition and contextual verbal learning was accounted for, whereas in the AD group the addition of executive measures did not add to prediction of variance in CVLT-II learning. These findings suggest that deficits in executive functions play a vital role in learning impairments in patients with PDD; however, for AD patients, learning difficulties appear relatively independent of executive dysfunction.

Background

Electronic clinical decision support (CDS) is increasingly establishing its role in evidence-based clinical practice. Considerable evidence supports its enhancement of efficiency in e-Prescribing, but some controversy remains. This study evaluated the practicality and identified the perceived benefits of, and barriers to, its future adoption in the West of Ireland.

Methods

This cross sectional study was carried out by means of a 27-part questionnaire sent to 262 registered general practitioners in Counties Galway, Mayo and Roscommon. The survey domains encompassed general information of individual's practice, current use of CDS and the practitioner's attitudes towards adoption of CDS-eP. Descriptive and inferential analyses were performed to analyse the data collected.

Results

The overall response rate was 37%. Nearly 92% of respondents employed electronic medical records in their practice. The majority acknowledged the value of electronic CDS in improving prescribing quality (71%) and reducing prescribing errors (84%). Despite a high degree of unfamiliarity (73%), the practitioners were open to the use of CDS-eP (94%) and willing to invest greater resources for its implementation (62%). Lack of a strategic implementation plan (78%) is the main perceived barrier to the incorporation of CDS-eP into clinical practice, followed by i) lack of financial incentives (70%), ii) lack of standardized product software (61%), iii) high sensitivity of drug-drug interaction or medication allergy markers (46%), iv) concern about overriding physicians' prescribing decisions(44%) and v) lack of convincing evidence on the systems' effectiveness (22%).

Conclusions

Despite favourable attitudes towards the adoption of CDS-eP, multiple perceived barriers impede its incorporation into clinical practice. These merit further exploration, taking into consideration the structure of the Irish primary health care system, before CDS-eP can be recommended for routine clinical use in the West of Ireland.

Background

Few comparative studies exist of metabolic brain changes among neurodegenerative illnesses. We compared brain metabolic abnormalities in Alzheimer’s disease (AD) and in Parkinson’s disease with dementia (PDD) as measured by proton magnetic resonance spectroscopy (MRS).

Methods

Twelve patients with idiopathic PDD, 22 patients with probable mild AD, and 61 healthy older controls underwent posterior cingulate MRS.

Results

Patients with AD showed reduced N-acetylaspartate (NAA)/creatine (Cr) (p <0.05) and increased choline (Cho)/Cr (p <0.05) and myo-Inositol (mI)/Cr (p <0.01) compared to controls. Patients with PDD showed reduced NAA/Cr (p <0.05) and glutamate (Glu)/Cr (p <0.01) compared to controls. There was reduced Glu/Cr in PDD compared to AD (p <0.01).

Conclusion

Patients with AD and patients with PDD showed distinct brain metabolic MRS profiles. Findings suggest that comparison of brain MRS profiles across dementias provides useful direction for future study.

Adeno-associated virus serotype 2 (AAV-2) has been developed as a gene therapy vector. Antibody and cell-mediated immune responses to AAV-2 or AAV-2-transfected cells may confound the therapeutic use of such vectors in clinical practice. In one of the most detailed examinations of AAV-2 immunity in humans to date, cell-mediated and humoral immune responses to AAV-2 were characterized from a panel of healthy blood donors. The extent of AAV-2-specific antibody in humans was determined by examination of circulating AAV-2-specific total IgG levels in plasma from 45 normal donors. Forty-one donors were seropositive and responses were dominated by IgG1 and IgG2 subclasses. Conversely, AAV-2-specific IgG3 levels were consistently low in all donors. Cell-mediated immune recall responses were detectable in nearly half the population studied. In vitro restimulation with AAV-2 of peripheral blood mononuclear cell cultures from 16 donors elicited gamma interferon (IFN-γ) (ten donors), interleukin-10 (IL-10) (eight donors) and interleukin-13 (IL-13) (four donors) responses. Using a series of overlapping peptides derived from the sequence of the VP1 viral capsid protein, a total of 59 candidate T-cell epitopes were identified. Human leukocyte antigen characterization of donors revealed that the population studied included diverse haplotypes, but that at least 17 epitopes were recognized by multiple donors and could be regarded as immunodominant. These data indicate that robust immunological memory to AAV-2 is established. The diversity of sequences recognized suggests that attempts to modify the AAV-2 capsid, as a strategy to avoid confounding immunity, will not be feasible.