Adaptive Radiation Therapy for Intact Thymoma: An Illustrative Report.

BACKGROUND/AIM: Adaptive radiation therapy (ART) is a technique capable of reducing radiation dose to normal tissue without compromising local control. For potentially resectable thymoma, induction therapy is standard of care. Because large disease volume is common in this context, ART has been suggested to reduce toxicity from induction chemoradiation. This has not been previously illustrated in the literature. CASE REPORT: A 38-year-old man with initially unresectable thymoma was treated with induction chemoradiation including cisplatin and etoposide. He received 45 Gy in 25 fractions and ART was utilized to shrink the radiotherapy field for the final 10 fractions. RESULTS: Thymectomy showed Masaoka stage III disease with negative margins. He experienced no treatment-related toxicity and has no evidence of disease 8 years after diagnosis. CONCLUSION: Induction chemoradiotherapy with ART appears to be feasible, safe, and efficacious for locally advanced intact thymoma.

A Phase 2 Clinical Trial of SABR Followed by Immediate Vertebroplasty for Spine Metastases.

PURPOSE: To determine the pain response and prevention of vertebral compression fractures (VCFs) after single-fraction stereotactic ablative radiation therapy (SABR) in conjunction with immediate vertebroplasty for spine metastases. METHODS AND MATERIALS: Patients with localized spine metastases free from VCF associated with loss of vertebral height with a pain score ≥4 using the visual analog scale were enrolled. Spine SABR was performed with 20 Gy delivered to the gross disease and 14 Gy to the contiguous bone marrow in a single fraction. Immediate, prophylactic vertebroplasty was performed within 1 month after spine SABR. The primary endpoint was pain response at 3 months compared to the historical control with external beam radiation therapy from Radiation Therapy Oncology Group study 9714. Secondary endpoints included pain response at 1 month, duration of pain response, vertebroplasty rate, VCF rate, local control, and overall survival. RESULTS: Thirty-five patients were enrolled, of whom 29 were deemed eligible and underwent single-fraction spine SABR. Twenty-three of these patients subsequently underwent prophylactic vertebroplasty. The 3-month pain response was significantly improved compared to Radiation Therapy Oncology Group study 9714: 95% versus 51% (P < .0001). The local control with a median follow-up of 9.6 months was 92%. The freedom from VCF was 90% at 1 year. Spine SABR was well tolerated with no grade 2 or higher toxicities. A single patient with disease extending from the vertebral body into the spinal canal developed vertebroplasty-related myelopathy, which was corrected with surgery. CONCLUSIONS: Single-fraction SABR immediately followed by prophylactic vertebroplasty improves pain response compared with conventional radiation therapy while providing long-term pain control and structural stability of the treated spine. Vertebroplasty is well tolerated as a prophylactic measure in patients without loss of vertebral height after spine SABR. Pain response and VCF rates are similar to patients undergoing SABR alone. Thus, patients who would benefit most from the addition of vertebroplasty need to be further identified.

Metabolic syndrome is a sequela of radiation exposure in hypothalamic obesity among survivors of childhood brain tumors.

Survivors of childhood brain tumors may be at risk for early onset of metabolic syndrome, possibly secondary to surgery and/or radiation exposure. This study examines effects of radiation exposure to hypothalamus-pituitary-adrenal axis (HPA) on metabolic risk among survivors of childhood brain tumors. One hundred forty-two met inclusion criteria; 60 had tumor surgery plus radiation exposure (>1 Gray (Gy)) to HPA. The second subgroup of 82 subjects had surgery only and were not exposed to radiation. Both subgroups had survived for approximately 5 years at the time of study. All had clinical evaluation, vital signs, anthropometry, measurement of body composition by dual X-ray absorptiometry and fasting laboratory assays (metabolic panel, insulin, C-peptide, insulin-like growth factor-1, leptin and adiponectin). Body composition data for both subgroups was compared with the National Health and Nutrition Survey (NHANES) subgroup of similar age, gender and body mass index. Cranial surgery was associated with obesity of similar severity in both subgroups. However, survivors exposed to radiation to the HPA also had increased visceral fat mass and high prevalence of growth hormone deficiency and metabolic syndrome. Fat mass alone did not explain the prevalence of the metabolic syndrome in radiation exposure subgroup. Other factors such as growth hormone deficiency may have contributed to metabolic risk. We conclude that prevalence of metabolic syndrome among subjects exposed to hypothalamic radiation was higher than expected from hypothalamic obesity alone. Radiation exposure may exert untoward endocrinopathies due to HPA exposure that worsens metabolic risk. Early screening for metabolic syndrome in this population is indicated.

Virtual Bronchoscopy-Guided Treatment Planning to Map and Mitigate Radiation-Induced Airway Injury in Lung SAbR.

PURPOSE: Radiation injury to the bronchial tree is an important yet poorly understood potential side effect in lung stereotactic ablative radiation therapy (SAbR). We investigate the integration of virtual bronchoscopy in radiation therapy planning to quantify dosage to individual airways. We develop a risk model of airway collapse and develop treatment plans that reduce the risk of radiation-induced airway injury. METHODS AND MATERIALS: Pre- and post-SAbR diagnostic-quality computerized tomography (CT) scans were retrospectively collected from 26 lung cancer patients. From each scan, the bronchial tree was segmented using a virtual bronchoscopy system and registered deformably to the planning CT. Univariate and stepwise multivariate Cox regressions were performed, examining factors such as age, comorbidities, smoking pack years, airway diameter, and maximum point dosage (Dmax). Logistic regression was utilized to formulate a risk function of segmental collapse based on Dmax and diameter. The risk function was incorporated into the objective function along with clinical dosage volume constraints for planning target volume (PTV) and organs at risk (OARs). RESULTS: Univariate analysis showed that segmental diameter (P = .014) and Dmax (P = .007) were significantly correlated with airway segment collapse. Multivariate stepwise Cox regression showed that diameter (P = .015), Dmax (P < .0001), and pack/years of smoking (P = .02) were significant independent factors associated with collapse. Risk management-based plans enabled significant dosage reduction to individual airway segments while fulfilling clinical dosimetric objectives. CONCLUSION: To our knowledge, this is the first systematic investigation of functional avoidance in lung SAbR based on mapping and minimizing doses to individual bronchial segments. Our early results show that it is possible to substantially lower airway dosage. Such dosage reduction may potentially reduce the risk of radiation-induced airway injury, while satisfying clinically prescribed dosimetric objectives.

Correction: The bacterial community associated with the sheep gastrointestinal nematode parasite Haemonchus contortus.

[This corrects the article DOI: 10.1371/journal.pone.0192164.].

The bacterial community associated with the sheep gastrointestinal nematode parasite Haemonchus contortus.

Culture-independent methods were used to study the microbiota of adult worms, third-stage larvae and eggs, both in faeces and laid in vitro, of Haemonchus contortus, a nematode parasite of the abomasa of ruminants which is a major cause of production losses and ill-health. Bacteria were identified in eggs, the female reproductive tract and the gut of adult and third-stage larvae (L3). PCR amplification of 16S rRNA sequences, denaturing gradient gel electrophoresis (DGGE) and clone libraries were used to compare the composition of the microbial communities of the different life-cycle stages of the parasites, as well as parasites and their natural environments. The microbiomes of adult worms and L3 were different from those in the abomasum or faeces respectively. The H. contortus microbiota was mainly comprised of members of the phyla Proteobacteria, Firmicutes and Bacteroidetes. Bacteria were localised in the gut, inside eggs and within the uterus of adult female worms using the universal FISH Eub338 probe, which targets most bacteria, and were also seen in these tissues by light and transmission electron microscopy. Streptococcus/Lactococcus sp. were identified within the distal uterus with the probe Strc493. Sequences from the genera Weissella and Leuconostoc were found in all life-cycle stages, except eggs collected from faeces, in which most sequences belonged to Clostridium sp. Bacteria affiliated with Weissella/Leuconostoc were identified in both PCR-DGGE short sequences and clone libraries of nearly full length 16S rRNA bacterial sequences in all life-cycle stages and subsequently visualised in eggs by fluorescent in situ hybridisation (FISH) with group-specific probes. This strongly suggests they are vertically transmitted endosymbionts. As this study was carried out on a parasite strain which has been maintained in the laboratory, other field isolates will need to be examined to establish whether these bacteria are more widely dispersed and have potential as targets to control H. contortus infections.

Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.

Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland.

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus.

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD(+)/H or NADP(+)/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD(+)/H than with NADP(+)/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.

Dosimetric and motion analysis of margin-intensive therapy by stereotactic ablative radiotherapy for resectable pancreatic cancer.

BACKGROUND: The retroperitoneal margin is a common site of positive surgical margins in patients with resectable pancreatic cancer. Preoperative margin-intensive therapy (MIT) involves delivery of a single high dose of ablative radiotherapy (30 Gy) focused on this surgically inaccessible margin, utilizing stereotactic techniques in an effort to reduce local failure following surgery. In this study, we investigated the motion of regional organs at risk (OAR) utilizing 4DCT, evaluated the dosimetric effects of abdominal compression (AC) to reduce regional motion, and compared various planning techniques to optimize MIT. METHODS: 10 patients were evaluated with 4DCT scans. All 10 patients had scans using AC and seven of the 10 patients had scans both with and without AC. The peak respiratory abdominal organ and major vessel centroid excursion was measured. A "sub-GTV" region was defined by a radiation oncologist and surgical oncologist encompassing the retroperitoneal margin typically lateral and posterior to the superior mesenteric artery (SMA), and a 3-5 mm margin was added to constitute the PTV. Identical 3D non-coplanar SABR (3DSABR) plans were designed for the average compression and non-compression scans. Compression scans were planned with 3DSABR, coplanar IMRT (IMRT), and Cyberknife (CK) planning techniques. Dose volume analysis was undertaken for various endpoints, comparing OAR doses with and without AC and for different planning methods. RESULTS: The mean PTV size was 20.2 cm3. Regional vessel motion of the SMA, celiac trunk, and renal vessels was small (< 5 mm) and not significantly impacted by AC. Mean pancreatic motion was > 5 mm, so AC has been used in all patients enrolled thus far. AC did not significantly increase OAR dose including the stomach and traverse colon. There were several statistically significant differences in the doses to OARs as a function of the type of planning modality used. CONCLUSIONS: AC does not significantly reduce the limited motion of structures in close proximity to the MIT target and does not significantly increase the dose to OARs that can be displaced by the compression plate. The treatment planning techniques evaluated in this study have different advantages with no clearly superior method in our analysis. Dose to adjacent vessels may be reduced with 3DSABR or IMRT techniques, while conformality is increased with IMRT or CK.

Biodistribution and safety assessment of AAV2-GAD following intrasubthalamic injection in the rat.

BACKGROUND: The steps necessary to translate promising new biological therapies to the clinic are poorly documented. For gene therapy, there are unique aspects that need to be addressed in biodistribution studies. Notably, the spread of the vector beyond the intended target cells or tissue may result in persistent unwanted biological activity or unpredictable biological events; thus, it is critical to evaluate the risks associated with viral vector-mediated gene transfer prior to embarking on human clinical trials. METHODS: In the present study, we conducted a comprehensive assessment of vector biodistribution throughout the brain, blood and major organs of rats that had been injected via the subthalamic nucleus with recombinant adeno-associated virus (AAV) expressing glutamic acid decarboxylase (GAD). In addition, behavioral and histological analyses were also performed. RESULTS: AAV genomes were not detected in blood or cerebrospinal fluid, and did not disseminate to organs outside of the brain in the majority of animals. In the brain, an average of 97.3% of AAV2-GAD genomes were restricted to the area of the ipsilateral subthalamic nucleus (STN). There were no discernable effects of AAV2-GAD on general health, and a behavioral assessment of the animals did not reveal any alteration in general behavior, exploration, locomotion or motor symmetry. CONCLUSIONS: The present study met Food and Drug Administration requirements, in addition to efficacy and toxicity studies in rodents and nonhuman primates, to support and supplement a Phase II clinical trial invloving the gene transfer of AAV2-GAD to the human STN for the potential therapy of Parkinson's disease.

Essential role of cytoplasmic cdk5 and Prx2 in multiple ischemic injury models, in vivo.

Recent evidence suggests that abnormal activation of cyclin-dependent kinase 5 (cdk5) is a critical prodeath signal in stroke. However, the mechanism(s) by which cdk5 promotes death is unclear. Complicating the role of cdk5 are the observations that cdk5 can exist in multiple cellular regions and possess both prosurvival and prodeath characteristics. In particular, the critical role of cytoplasmic or nuclear cdk5 in neuronal jury, in vivo, is unclear. Therefore, we determined where cdk5 was activated in models of ischemia and how manipulation of cdk5 in differing compartments may affect neuronal death. Here, we show a critical function for cytoplasmic cdk5 in both focal and global models of stroke, in vivo. Cdk5 is activated in the cytoplasm and expression of DNcdk5 localized to the cytoplasm is protective. Importantly, we also demonstrate the antioxidant enzyme Prx2 (peroxiredoxin 2) as a critical cytoplasmic target of cdk5. In contrast, the role of cdk5 in the nucleus is context-dependent. Following focal ischemia, nuclear cdk5 is activated and functionally relevant while there is no evidence for such activation following global ischemia. Importantly, myocyte enhancer factor 2D (MEF2D), a previously described nuclear target of cdk5 in vitro, is also phosphorylated by cdk5 following focal ischemia. In addition, MEF2D expression in this paradigm ameliorates death. Together, our results address the critical issue of cdk5 activity compartmentalization, as well as define critical substrates for both cytoplasmic and nuclear cdk5 activity in adult models of stroke.

Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates.

Adeno-associated viral (AAV) vectors have become the primary delivery agent for somatic gene transfer into the central nervous system (CNS). To date, AAV-mediated gene delivery to the CNS is based on serotypes 1-9, with efficient gene transfer to neurons only-selective and widespread transduction of glial cells have not been observed. Recently, additional endogenous AAVs have been isolated from nonhuman primate tissues. In this study, transduction obtained with AAV serotypes bb2, cy5, rh20, rh39, and rh43 was compared to that obtained with AAV8, another nonhuman primate isolate previously shown to perform well in mammalian brain. Titer-matched vectors encoding the enhanced green fluorescent protein (EGFP) reporter, driven by the constitutive CAG promoter, were injected into the hippocampus, striatum, or substantia nigra (SN) of adult rats. More widespread neuronal transduction was observed following infusion of cy5, rh20, and rh39 than observed with AAV8. Of interest, preferential transduction of astrocytes was observed with rh43. To optimize glial transduction, vector stocks driven by cell-specific promoters were generated-widespread and targeted transduction of astrocytes and oligodendrocytes was observed using rh43 and AAV8, driven by the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) promoters, expanding the utility of AAV for modeling and treating diseases involving glial cell pathology.

Regulation of the postnatal development of dopamine neurons of the substantia nigra in vivo by Akt/protein kinase B.

Following mitosis, specification and migration during embryogenesis, dopamine neurons of the mesencephalon undergo a postnatal naturally occurring cell death event that determines their final adult number, and a period of axonal growth that determines pattern and extent of target contacts. While a number of neurotrophic factors have been suggested to regulate these developmental events, little is known, especially in vivo, of the cell signaling pathways that mediate these effects. We have examined the possible role of Akt/Protein Kinase B by transduction of these neurons in vivo with adeno-associated viral vectors to express either a constitutively active or a dominant negative form of Akt/protein kinase B. We find that Akt regulates multiple features of the postnatal development of these neurons, including the magnitude of the apoptotic developmental cell death event, neuron size, and the extent of target innervation of the striatum. Given the diversity and magnitude of its effects, the regulation of the development of these neurons by Akt may have implications for the many psychiatric and neurologic diseases in which these neurons may play a role.

Neuropeptide Y overexpression using recombinant adeno-associated viral vectors.

Gene therapy may represent a promising alternative treatment of epileptic patients who are resistant to conventional anti-epileptic drugs. Among the various approaches for the application of gene therapy in the treatment of CNS disorders, recombinant adeno-associated viral (AAV) vectors have been most widely used. Preclinical studies using a selection of "therapeutic" genes injected into the rodent brain to correct the compromised balance between inhibitory and excitatory transmission in epilepsy, showed significant reduction of seizures and inhibition of epileptogenesis. In particular, transduction of neuropeptide genes, such as galanin and neuropeptide Y (NPY) in specific brain areas in experimental models of seizures resulted in significant anticonvulsant effects. Recent findings showed a long-lasting NPY over-expression in the rat hippocampus by local application of recombinant AAV vectors associated with reduced generalization of seizures, delayed kindling epileptogenesis, and strong reduction of chronic spontaneous seizures. These results establish a proof-of-principle evidence of the efficacy of gene therapy as anticonvulsant treatment. Additional investigations are required to address safety concerns and possible side effects in more detail.

Neuropeptide Y gene therapy decreases chronic spontaneous seizures in a rat model of temporal lobe epilepsy.

Temporal lobe epilepsy remains amongst the most common and drug refractory of neurological disorders. Gene therapy may provide a realistic therapeutic approach alternative to surgery for intractable focal epilepsies. To test this hypothesis, we applied here a gene therapy approach, using a recombinant adeno-associated viral (rAAV) vector expressing the human neuropeptide Y (NPY) gene, to a progressive and spontaneous seizure model of temporal lobe epilepsy induced by electrical stimulation of the temporal pole of the hippocampus, which replicates many features of the human condition. rAAV-NPY or a control vector lacking the expression cassette (rAAV-Empty) was delivered into the epileptic rat hippocampi at an early progressive stage of the disease. Chronic epileptic rats were video-EEG monitored to establish pre-injection baseline recordings of spontaneous seizures and the effect of rAAV-NPY versus rAAV-Empty vector injection. Both non-injected stimulated controls and rAAV-empty injected rats showed a similar progressive increase of spontaneous seizure frequency consistent with epileptogenesis. The delivery of rAAV-NPY in epileptic rat brain leads to a remarkable decrease in the progression of seizures as compared to both control groups and this effect was correlated with the NPY over-expression in the hippocampus. Moreover, spontaneous seizure frequency was significantly reduced in 40% of treated animals as compared to their pre-injection baseline. Our data show that this gene therapy strategy decreases spontaneous seizures and suppresses their progression in chronic epileptic rats, thus representing a promising new therapeutic strategy.

HSP70 and constitutively active HSF1 mediate protection against CDCrel-1-mediated toxicity.

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.

Antiapoptotic and trophic effects of dominant-negative forms of dual leucine zipper kinase in dopamine neurons of the substantia nigra in vivo.

There is extensive evidence that the mitogen-activated protein kinase (MAPK) signaling cascade mediates programmed cell death in neurons. However, current evidence that the mixed linage kinases (MLKs), upstream in this cascade, mediate cell death is based, in the in vivo context, entirely on pharmacological approaches. The compounds used in these studies have neither complete specificity nor selectivity among these kinases. Therefore, to better address the molecular specificity of the MLKs in mediating neuron death, we used dominant-negative constructs delivered by AAV (adenoassociated virus) vector transfer. We assessed effects in a neurotoxin model of parkinsonism, in which neuroprotection by pharmacologic MLK inhibition has been reported. We find that two dominant-negative forms of dual leucine zipper kinase (DLK) inhibit apoptosis and enhance long-term survival of dopamine neurons, but a dominant negative of MLK3 does not. Interestingly, the kinase-dead form of DLK not only blocks apoptosis but also has trophic effects on dopamine neurons. Although the MAPK cascade activates a number of downstream cell death mediators, we find that inhibition of DLK correlates closely with blockade of phosphorylation of c-jun and prevention of cell death. We conclude that DLK acts primarily through c-jun phosphorylation to mediate cell death in this model.

The Parkinson's disease gene DJ-1 is also a key regulator of stroke-induced damage.

Recent evidence has indicated that common mechanisms play roles among multiple neurological diseases. However, the specifics of these pathways are not completely understood. Stroke is caused by the interruption of blood flow to the brain, and cumulative evidence supports the critical role of oxidative stress in the ensuing neuronal death process. DJ-1 (PARK7) has been identified as the gene linked to early-onset familial Parkinson's disease. Currently, our work also shows that DJ-1 is central to death in both in Vitro and in Vivo models of stroke. Loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia, whereas expression of DJ-1 can reverse this sensitivity and indeed provide further protection. Importantly, DJ-1 expression decreases markers of oxidative stress after stroke insult in Vivo, suggesting that DJ-1 protects through alleviation of oxidative stress. Consistent with this finding, we demonstrate the essential role of the oxidation-sensitive cysteine-106 residue in the neuroprotective activity of DJ-1 after stroke. Our work provides an important example of how a gene seemingly specific for one disease, in this case Parkinson's disease, also appears to be central in other neuropathological conditions such as stroke. It also highlights the important commonalities among differing neuropathologies.

Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Abeta levels.

BACKGROUND: Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-beta peptide (Abeta) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Abeta42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Abeta40, the more prevalent Abeta peptide secreted by cells and a major component of cerebral Abeta deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Abeta42 and Abeta40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. RESULTS: Adeno-associated viral (AAV) vectors encoding BRI-Abeta cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Abeta peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Abeta40 and AAV-BRI-Abeta42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Abeta peptides. BRI-Abeta42 and the combination of BRI-Abeta40+42 overexpression resulted in elevated levels of detergent-insoluble Abeta. No significant increase in detergent-insoluble Abeta was seen in the rats expressing APPsw or BRI-Abeta40. No pathological features were noted in any rats, except the AAV-BRI-Abeta42 rats which showed focal, amorphous, Thioflavin-negative Abeta42 deposits. CONCLUSION: The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Abeta42 alone is sufficient to initiate Abeta deposition, both Abeta40 and Abeta42 may contribute to cognitive deficits.