Transient suppression of hepatocellular replication in the mouse liver following transduction with recombinant adeno-associated virus.

Recombinant vectors based on adeno-associated virus (AAV) are proving to be powerful tools for genetic manipulation of the liver, for both discovery and therapeutic purposes. The system can be used to deliver transgene cassettes for expression or, alternatively, DNA templates for genome editing via homologous recombination. The replicative state of target cells is known to influence the efficiency of these processes and knowledge of the host-vector interactions involved is required for optimally effective vector deployment. Here we show, for the first time in vivo, that in addition to the known effects of hepatocellular replication on AAV-mediated gene transfer, the vector itself exerts a potent, albeit transient suppressive effect on cell cycle progression that is relieved on a time course that correlates with the known rate of clearance of input single-stranded vector DNA. This finding requires further mechanistic investigation, delineates an excellent model system for such studies and further deepens our insight into the complexity of interactions between AAV vectors and the cell cycle in a clinically promising target tissue.

Massive splenomegaly correlates with malignancy: 180 cases of splenic littoral cell tumors in the world literature.

Littoral cell tumors (LCT) are rare primary splenic neoplasms, unique for their morphologic and immunolabeling features resembling the endothelial littoral cells lining the sinusoids of the red pulp. They include the more common and typically benign littoral cell angioma, as well as the less common, potentially malignant, littoral cell hemangioendothelioma (LCHE) and the aggressive littoral cell angiosarcoma (LCAS). The most common presentation of these neoplasms is splenomegaly, and diagnosis is made histologically following biopsy or resection. To better understand these tumors, a comprehensive, international literature search was performed. Patient and tumor data, including presenting symptoms, comorbid cancers, immunosuppressive states, splenic mass and tumor size were analyzed. Massive splenomegaly (≥ 1500 g) following splenic resection, which correlates with a splenic length of 20 cm preoperatively, was found to be significantly associated with the presence of malignancy in the LCT (P<0.05).

Comparison of gene transfer to the murine liver following intraperitoneal and intraportal delivery of hepatotropic AAV pseudo-serotypes.

Adeno-associated virus (AAV) vectors are highly efficient for liver-targeted gene delivery in murine models and show promise in early phase human clinical trials. This efficiency is capsid-dependent and was only achieved after discovery that the AAV2 vector genome could be trans-encapsidated into the capsids of other AAV serotypes. This confers novel host-vector biology and target tissue tropism. Optimal exploitation of the growing number of AAV vector pseudo-serotypes, however, requires detailed context-dependent characterisation of transduction performance. In this study, we compared the pattern and efficiency of gene delivery to the adult mouse liver following intraportal and intraperitoneal injection of vectors pseudo-serotyped with known hepatotropic capsids from AAV type 7, 8, 9 and rhesus 10. Vectors pseudo-serotyped with these hepatotropic capsids proved relatively efficient irrespective of administration route, with higher transgene expression in males despite equivalent vector genome delivery in females. Transgene expression was predominantly centrilobular in contrast to the AAV2 capsid, which gave a periportal pattern of expression. Most intriguingly, vector genome performance appeared to be delivery route-dependent, consistent with the possibility of in vivo capsid modification. These data not only inform the experimental use of AAV vectors, but also provide insight into novel aspects of host-vector biology requiring further focused analysis.

AAV-encoded OTC activity persisting to adulthood following delivery to newborn spf(ash) mice is insufficient to prevent shRNA-induced hyperammonaemia.

Urea cycle defects presenting in the neonatal period with hyperammonaemia are associated with high morbidity and mortality, and necessitate liver transplantation for long-term management. Gene therapy is therefore an attractive possibility, with vectors based on adeno-associated virus (rAAV) currently showing exciting promise in liver-targeted clinical trials in adults. Successful use of rAAV vectors in infants, however, is more challenging as episomal rAAV genomes will be lost from proliferating hepatocytes during liver growth, leaving stable transgene expression dependent on the subset of vector genomes that undergo genomic integration. To explore this challenge, we exploited the partially ornithine transcarbamylase (OTC)-deficient spf(ash) mouse model and small hairpin RNA-mediated knockdown of residual endogenous OTC enzyme activity in adult mice that had received neonatal treatment with an OTC-encoding rAAV. This leaves mice reliant on vector-encoded OTC activity that has persisted from the newborn period. Despite stable transduction in approximately 8% of hepatocytes and residual vector-encoded OTC activity of up to 33% of wild-type, well above endogenous spf(ash) levels (5-7%), mice were not protected from hyperammonaemia. These data show that the distribution of OTC activity within the liver is critical and that rAAV vector re-delivery after early neonatal treatment is likely to be necessary for stable control of hyperammonaemia into adulthood.

A rapid, controlled-environment seedling root screen for wheat correlates well with rooting depths at vegetative, but not reproductive, stages at two field sites.

BACKGROUND AND AIMS: Root length and depth determine capture of water and nutrients by plants, and are targets for crop improvement. Here we assess a controlled-environment wheat seedling screen to determine speed, repeatability and relatedness to performance of young and adult plants in the field. METHODS: Recombinant inbred lines (RILs) and diverse genotypes were grown in rolled, moist germination paper in growth cabinets, and primary root number and length were measured when leaf 1 or 2 were fully expanded. For comparison, plants were grown in the field and root systems were harvested at the two-leaf stage with either a shovel or a soil core. From about the four-leaf stage, roots were extracted with a steel coring tube only, placed directly over the plant and pushed to the required depth with a hydraulic ram attached to a tractor. KEY RESULTS: In growth cabinets, repeatability was greatest (r = 0.8, P < 0.01) when the paper was maintained moist and seed weight, pathogens and germination times were controlled. Scanned total root length (slow) was strongly correlated (r = 0.7, P < 0.01) with length of the two longest seminal axile roots measured with a ruler (fast), such that 100-200 genotypes were measured per day. Correlation to field-grown roots at two sites at two leaves was positive and significant within the RILs and cultivars (r = 0.6, P = 0.01), and at one of the two sites at the five-leaf stage within the RILs (r = 0.8, P = 0.05). Measurements made in the field with a shovel or extracted soil cores were fast (5 min per core) and had significant positive correlations to scanner measurements after root washing and cleaning (>2 h per core). Field measurements at two- and five-leaf stages did not correlate with root depth at flowering. CONCLUSIONS: The seedling screen was fast, repeatable and reliable for selecting lines with greater total root length in the young vegetative phase in the field. Lack of significant correlation with reproductive stage root system depth at the field sites used in this study reflected factors not captured in the screen such as time, soil properties, climate variation and plant phenology.

In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver.

Mutant proteins have the potential to exert dominant-negative effects that might limit the therapeutic efficacy of their wild-type counterparts after gene transfer. For ornithine transcarbamylase (OTC) deficiency, in vitro studies have suggested the presence of dominant-negative effects, however, supporting in vivo studies have not been conducted. In this study, we exploited the capacity of recombinant adeno-associated virus (rAAV) 2/8 vectors to deliver transgenes to the mouse liver with high efficiency to determine whether expression of selected OTC mutant proteins exert inhibitory effects on endogenous wild-type OTC enzymatic activity. Using site-directed mutagenesis we constructed three OTC mutants with a theoretical or reported in vitro capacity to exert dominant-negative effects, and delivered these to the liver using rAAV2/8. Each mutation had been earlier identified in patients with OTC deficiency. Treated mice showed no increase in urinary orotic acid levels or reduction in OTC activity despite supra-physiological expression of the mutant proteins, consistent with an absence of dominant-negative effects. These data have important implications for the development of gene therapy strategies for OTC deficiency and validate a model system in which potential dominant-negative effects of specific mutations in prospective patients can be examined empirically before gene therapy.

Potential of AAV vectors in the treatment of metabolic disease.

Inborn errors of metabolism are collectively common, frequently severe and in many instances difficult or impossible to treat. Accordingly, there is a compelling need to explore novel therapeutic modalities, including gene therapy, and examine multiple phenotypes where the risks of experimental therapy are outweighed by potential benefits to trial participants. Among available gene delivery systems recombinant AAV shows special promise for the treatment of metabolic disease given the unprecedented efficiencies achieved in transducing key target tissues, such as liver and muscle, in small animal models. To date over 30 metabolic disease phenotypes have been investigated in small animal studies with complete phenotype correction being achieved in a substantial proportion. Achieving adequately widespread transduction within the central nervous system, however, remains a major challenge, and will be critical to realization of the therapeutic potential of gene therapy for many of the most clinically troubling metabolic disease phenotypes. Despite the relatively low immunogenicity of AAV vectors, immune responses are also emerging as a factor requiring special attention as efforts accelerate toward human clinical translation. Four metabolic disease phenotypes have reached phase I or I/II trials with one, targeting lipoprotein lipase deficiency, showing exciting early evidence of efficacy.

Foliar temperature tolerance of temperate and tropical evergreen rain forest trees of Australia.

Australian rain forests extend from tropical climates in the north to temperate climates in the south, providing an opportunity to investigate physiological responses to temperature of both temperate and tropical species within the same forest type. Eight, rain forest canopy tree species were selected to cover the 33 degrees latitudinal range of rain forests in eastern Australia. Temperature tolerance was measured in 6-year-old plants grown in a common environment, by exposing leaves to a series of high temperatures during late summer and a series of freezing temperatures during midwinter. Damage was evaluated based on chlorophyll fluorescence measurements made 2 h after exposure and by visual assessment of leaf damage made a week after exposure. Leaves of the tropical species were more heat tolerant and less frost tolerant than leaves of the temperate species, which is consistent with their climate distributions. In contrast, the temperature tolerance of the photosynthetic apparatus was unrelated to climate in a species' native habitat. However, the tropical species underwent significant photoinhibition during winter. All species maintained the integrity of the photosynthetic apparatus and avoided tissue damage over a similar span of temperatures (about 60 degrees C), reflecting the similar annual temperature ranges in Australia's temperate and tropical rain forests. Chlorophyll fluorescence measurements and visual assessment of leaf damage provided different estimates of the absolute and relative temperature tolerances of the species, thus emphasizing the importance of a direct assessment of tissue damage for determining a species' temperature tolerance.

Riparian reforestation: are there changes in soil carbon and soil microbial communities?

Reforestation of pastures in riparian zones has the potential to decrease nutrient runoff into waterways, provide both terrestrial and aquatic habitat, and help mitigate climate change by sequestering carbon (C). Soil microbes can play an important role in the soil C cycle, but are rarely investigated in studies on C sequestration. We surveyed a chronosequence (0-23years) of mixed-species plantings in riparian zones to investigate belowground (chemical and biological) responses to reforestation. For each planting, an adjacent pasture was surveyed to account for differences in soil type and land-use history among plantings. Two remnant woodlands were included in the survey as indicators of future potential of plantings. Both remnant woodlands had significantly higher soil organic C (SOC) content compared with their adjacent pastures. However, there was no clear trend in SOC content among plantings with time since reforestation. The substantial variability in SOC sequestration among plantings was possibly driven by differences in soil moisture among plantings and the inherent variability of SOC content among reference pastures adjacent to plantings. Soil microbial phospholipid fatty acids (PLFA, an indicator of microbial biomass) and activities of decomposition enzymes (ß-glucosidase and polyphenol oxidase) did not show a clear trend with increasing planting age. Despite this, there were positive correlations between total SOC concentration and microbial indicators (total PLFA, fungal PLFA, bacterial PLFA and activities of decomposition enzymes) across all sites. The soil microbial community compositions (explored using PLFA markers) of older plantings were similar to those of remnant woodlands. There was a positive correlation between the soil carbon:nitrogen (C:N) and fungal:bacterial (F:B) ratios. These data indicate that in order to maximise SOC sequestration, we need to take into account not only C inputs, but the microbial processes that regulate SOC cycling as well.

Vive la résistance: reviving resistance for 21st century conservation.

Confronted with increasing anthropogenic change, conservation in the 21st century requires a sound understanding of how ecological systems change during disturbance. We highlight the benefits of recognizing two distinct components of change in an ecological unit (i.e., ecosystem, community, population): 'resistance', the ability to withstand disturbance; and 'resilience', the capacity to recover following disturbance. By adopting a 'resistance-resilience' framework, important insights for conservation can be gained into: (i) the key role of resistance in response to persistent disturbance, (ii) the intrinsic attributes of an ecological unit associated with resistance and resilience, (iii) the extrinsic environmental factors that influence resistance and resilience, (iv) mechanisms that confer resistance and resilience, (v) the post-disturbance status of an ecological unit, (vi) the nature of long-term ecological changes, and (vii) policy-relevant ways of communicating the ecological impacts of disturbance processes.

Do temperate rainforest trees have a greater ability to acclimate to changing temperatures than tropical rainforest trees?

• Photosynthetic responses to acclimation temperature were investigated in seedlings of eight Australian rainforest tree species. Australian rainforests extend over 33° of latitude, providing an opportunity to compare temperature responses of temperate and tropical species. • Net photosynthesis was measured in leaves developed under a constant (22°C : 14°C) or fluctuating (17°C : 9°C-27°C : 19°C) day/night temperature regime. These leaves were then subjected to a series of constant temperature regimes and net photosynthesis was measured 14 d after acclimation to each new regime. • Acclimation potential was not affected by the contrasting temperature regimes. The temperate species showed at least 80% of maximum net photosynthesis over a larger span of acclimation temperature than the tropical species. • The lack of an effect of the contrasting temperature regimes on acclimation potential may reflect either that adjustments were unnecessary for temperate species, which already have broad photosynthetic responses to temperature, and tropical species were incapable of adjustments, or that in general species respond to the mean temperature regime and not to the amount of fluctuation in the regime. The higher acclimation potential shown by the temperate species is consistent with the larger seasonal and day-to-day variation in temperature of the temperate climate compared with the tropical climate.

The antigenicity of the chicken anaemia virus protein VP3 (Apoptin).

Chicken anaemia virus protein VP3 (Apoptin) was cloned and expressed as a recombinant protein and evaluated for its suitability as a serodiagnostic reagent. VP3 was expressed as a fusion protein either with glutathione S -transferase or with a six-histidine tag. Both recombinant proteins reacted specifically with anti-VP3 monoclonal antibodies and with serum from vaccinated chickens by Western blot and by enzyme-linked immunosorbent assay (ELISA). However, when testing sera from birds of different ages and genetic backgrounds, high non-specific reactions were evident and false positives were observed, especially in older birds. This suggests that VP3 is poorly immunogenic during infection and low antibody concentrations are masked by non-specific reactions. Thus, VP3 is not suitable for use as antigen in ELISAs.

Psychosocial factors associated with condom use among African-American drug abusers in treatment.

Although strategies for decreasing injection drug use have met with moderate success, efforts to decrease high-risk sexual behaviors have been less successful. Because condom use reduces HIV transmission, it is critically important to identify the attitudinal, emotional, and behavioral factors associated with using condoms. This study evaluated the relationship between condom use and various psychological and behavioral variables among heterosexual, African-American, cocaine-dependent men within the context of the AIDS Risk Reduction Model (ARRM). Subjects who used condoms (n = 52) reported significantly higher levels of self-efficacy, condom use skills, and sexual communication with partners than non-users (n = 84). However, the groups did not differ in perceived susceptibility, anxiety concerning HIV transmission, response efficacy, or knowledge regarding HIV. These findings suggest that future interventions focus on enhancing self-efficacy and condom use skills, as well as eroticizing condom use.

PIP: While strategies to decrease the prevalence and frequency of injection drug use have proved to be moderately successful, only minimal success has been achieved in getting IV drug users to reduce the amount of high-risk sex they have. It is common among heavy drug users, however, to be promiscuous and exchange sex for drugs and money. Injecting drug users rarely use condoms to reduce their risk of HIV transmission, even though it has been found that more than 70% of AIDS cases among heterosexuals may be attributed to sexual contact with drug users. Since consistent condom use can reduce the risk of HIV transmission, it is extremely important to identify the attitudinal, emotional, and behavioral factors associated with their use. This study was therefore conducted to evaluate the relationship between condom use and various psychological and behavioral variables among heterosexual, African-American, cocaine-dependent men within the context of the AIDS Risk Reduction Model. 136 subjects of mean age 35.5 years and an average education of 13.1 years were studied. 112 subjects smoked cocaine and 24 injected it. The 52 subjects who used condoms reported significantly higher levels of self-efficacy, condom use skills, and sexual communication with partners than the 84 non-users. The groups did not, however, differ in perceived susceptibility, anxiety concerning HIV transmission, response efficacy, or knowledge regarding HIV. Study findings suggest that future interventions focus on enhancing self-efficacy and condom use skills, as well as eroticizing condom use.

Outcome of psychoeducation for HIV risk reduction.

Our objectives were to assess the effects of a psychoeducational (PE) program designed to reduce HIV risk behaviors in recovering drug abusers and to evaluate mediating variables associated with risk reduction as described by the AIDS Risk Reduction Model (ARRM). Consecutive admissions to a Department of Veterans Affairs drug dependence inpatient treatment program (n = 152) were randomly assigned to PE or a standard information (INFO) condition. PE involved a 6-hour small group intervention designed to enhance knowledge and attitudes regarding HIV prevention, improve skills in condom use and needle sterilization, and modify high-risk sex- and drug-related behaviors. The INFO condition involved presentation of audiovisual and printed HIV prevention material with similar content. Following intervention, PE subjects showed significantly enhanced self-efficacy, condom use skills, and sexual communication skills relative to the INFO group. At 3-month follow-up, the PE group showed significantly greater reductions on some measures of sexual HIV risk behaviors relative to the INFO group. Hypotheses derived from the ARRM regarding presumed relationships between positive changes in mediating variables (e.g., self-efficacy and sexual communication) and ultimate outcome variables (e.g., condom use) were supported.

PIP: The objectives were to assess the effects of a psychoeducational (PE) program designed to reduce HIV risk behaviors in recovering drug abusers and to evaluate mediating variables associated with risk reduction as described by the AIDS Risk Reduction Model (ARRM). African-American male participants with no evidence of HIV seropositivity were recruited over a 10-month period from among 235 consecutive admissions to the Department of Veteran's Affair Drug Dependence Treatment Program (DDTP). 176 subjects fulfilled eligibility criteria. 152 consecutive admissions to DDTP were randomly assigned to either or standard information (INFO) groups of equal size. PE involved a 6-hour small group intervention to enhance knowledge regarding HIV prevention, improve condom use and needle sterilization skills, and modify high-risk sex- and drug-related behaviors. The INFO condition involved presentation of HIV prevention materials with similar content. Subjects were an average of 35.5 years old with 13.06 years old with 13.06 years of education, and an estimated IQ of 105.4 Overall, subjects reported a significant reduction in high-risk behaviors at 3 months compared to preintervention levels (p .0001). 75% of the subjects in the PE group reported higher sexual risk behaviors before the intervention vs. 32% at 3 months (p .0001). Also, 75% of INFO subjects were at higher risk pretreatment, which decreased to 48% at 3 months (p .01). There was a trend toward greater risk reduction in the PE group. 25 (62.5%) of the 40 PE subjects at higher risk prior to the intervention were at lower risk at the 3-month follow-up. In the INFO group, 51.2% of the preintervention higher risk subjects were at lower risk 3 months postintervention. Among the 107 subjects completing the 3-month follow-up, significantly fewer reported more than one partner, from 75.5% preintervention to 50.5% at 3 months (p = .0001). 75.5% of PE subjects reported more than one sexual partner preintervention vs. 47.5% at 3 months (binomial p = .0001). In the INFO group, this declined from 75.9% to 59.3%, which was without statistical significance.

Changing character of subacute sclerosing panencephalitis in the United States.

We analyzed National Registry data from 575 patients with subacute sclerosing panencephalitis (SSPE) in the United States to assess changes in patient characteristics and SSPE epidemiology. Racial proportions have changed in recent years with an increasing number of Hispanic patients reported in relation to a constant black:white ratio; however, the male:female ratio of approximately 2:1 has remained. The most striking feature of the data is the rapid decline in SSPE incidence. Corresponding to this decrease is an increase in the proportion of cases following measles vaccination. There also is a shorter incubation period for SSPE following vaccination than after measles infection.

Photosynthetic responses to vapour pressure deficit in temperate and tropical evergreen rainforest trees of Australia.

Rainforests occur in high precipitation areas of eastern Australia, along a gradient in seasonality of precipitation, ranging from a summer dry season in the temperate south to a winter dry season in the tropical north. The response of net photosynthesis to increasing vapour pressure deficit (VPD) was measured in a range of Australian rainforest trees from different latitudes to investigate possible differences in their response to atmospheric drought. Plants were grown in glasshouses under ambient or low VPD to determine the effect of growth VPD on the photosynthetic response. Temperate species, which experience low summer precipitation, were found to maintain maximum net photosynthesis over the measurement range of VPD (0.5-1.9 kPa). In contrast, the tropical species from climates with high summer precipitation showed large reductions in net photosynthesis with increasing VPD. Temperate species showed higher intrinsic water-use efficiencies under low VPD than the tropical species, whereas their efficiencies were similar under high VPD. Growing plants under a low VPD had little effect on either the photosynthetic response to VPD or the intrinsic water-use efficiency of the species. These different responses of gas exchange to VPD shown by the tropical and temperate rainforest species may reflect different strategies to maximise productivity in their respective climates.

Cytodiagnosis of breast disease using a liquid suspension medium.

Fine-needle aspiration cytology specimens from 187 breast lesions were processed using conventional slide cytology. Results were compared with those from 201 lesions prepared using a liquid suspension medium (Cytospin) for specimen collection, to assess the clinical utility of each technique. The frequency of diagnostic groups was similar for each method. No significant difference was found in sensitivity (conventional slide cytology 86 per cent versus Cytospin 82 per cent), specificity (72 versus 82 per cent) or diagnostic accuracy (88 versus 91 per cent). The Cytospin technique compares favourably with conventional slide cytology and can be recommended for ease of use in breast cytodiagnosis.

'To bead or not to bead?'.

In subcuticular wound closure, the use of anchorage beads or knots to maintain opposition of wound edges is a common practice, but can lead to complications. One hundred and one abdominal subcuticular wound closures (44 vertical and 57 transverse) were prospectively studied and assessment of wound swelling was made by measuring the length of exposed suture at both ends of the wound, on successive days postoperatively. Results showed progressive reduction in the exposed suture length. We would suggest that if anchorage beads are used with subcuticular sutures then they should not be pulled tight as postoperative wound swelling will cause the beads to be drawn into the wound often resulting in a troublesome ulcer(s) at the end(s) of the wound.

Peroxisomal disease cell lines with cellular plasmalogen deficiency have impaired muscarinic cholinergic signal transduction activity and amyloid precursor protein secretion.

We tested whether alterations in membrane lipid composition associated with peroxisomal diseases affect muscarinic cholinergic signal transduction activity and amyloid precursor protein (APP) secretion in cultured human skin fibroblasts and Chinese hamster ovary (CHO) mutants. We found that in cell lines from patients with peroxisomal disorders where plasmalogen levels were low, the low-Km GTPase activity was not induced by carbachol, and APP secretion was reduced. This effect on signal transduction activity was not associated with decreased levels of the M1-muscarinic cholinergic receptor or its associated heterotrimeric G-protein. Specifically, this decrease was associated with a plasmalogen deficiency since a CHO cell line with only a deficit in plasmalogens was as severely affected as were generalized peroxisomal disorder cell lines. Thus, plasmalogens appear to be implicated in muscarinic cholinergic signal transduction and secretion of APP. These results provide new insights about the pathophysiology of peroxisomal diseases and may be relevant to Alzheimer's disease where reduced plasmalogen levels have been reported.