Mutation-specific reporter for optimization and enrichment of prime editing.

Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.

Nitrogen use efficiency and nitrous oxide emissions from five UK fertilised grasslands.

Intensification of grasslands is necessary to meet the increasing demand of livestock products. The application of nitrogen (N) on grasslands affects the N balance therefore the nitrogen use efficiency (NUE). Emissions of nitrous oxide (N2O) are produced due to N fertilisation and low NUE. These emissions depend on the type and rates of N applied. In this study we have compiled data from 5 UK N fertilised grassland sites (Crichton, Drayton, North Wyke, Hillsborough and Pwllpeiran) covering a range of soil types and climates. The experiments evaluated the effect of increasing rates of inorganic N fertiliser provided as ammonium nitrate (AN) or calcium ammonium nitrate (CAN). The following fertiliser strategies were also explored for a rate of 320 kg N ha-1: using the nitrification inhibitor dicyandiamide (DCD), changing to urea as an N source and splitting fertiliser applications. We measured N2O emissions for a full year in each experiment, as well as soil mineral N, climate data, pasture yield and N offtake. N2O emissions were greater at Crichton and North Wyke whereas Drayton, Hillsborough and Pwllpeiran had the smallest emissions. The resulting average emission factor (EF) of 1.12% total N applied showed a range of values for all the sites between 0.6 and 2.08%. NUE depended on the site and for an application rate of 320 kg N ha-1, N surplus was on average higher than 80 kg N ha-1, which is proposed as a maximum by the EU Nitrogen Expert Panel. N2O emissions tended to be lower when urea was applied instead of AN or CAN, and were particularly reduced when using urea with DCD. Finally, correlations between the factors studied showed that total N input was related to Nofftake and Nexcess; while cumulative emissions and EF were related to yield scaled emissions.

The contribution of cattle urine and dung to nitrous oxide emissions: Quantification of country specific emission factors and implications for national inventories.

Urine patches and dung pats from grazing livestock create hotspots for production and emission of the greenhouse gas, nitrous oxide (N2O), and represent a large proportion of total N2O emissions in many national agricultural greenhouse gas inventories. As such, there is much interest in developing country specific N2O emission factors (EFs) for excretal nitrogen (EF3, pasture, range and paddock) deposited during gazing. The aims of this study were to generate separate N2O emissions data for cattle derived urine and dung, to provide an evidence base for the generation of a country specific EF for the UK from this nitrogen source. The experiments were also designed to determine the effects of site and timing of application on emissions, and the efficacy of the nitrification inhibitor, dicyandiamide (DCD) on N2O losses. This co-ordinated set of 15 plot-scale, year-long field experiments using static chambers was conducted at five grassland sites, typical of the soil and climatic zones of grazed grassland in the UK. We show that the average urine and dung N2O EFs were 0.69% and 0.19%, respectively, resulting in a combined excretal N2O EF (EF3), of 0.49%, which is <25% of the IPCC default EF3 for excretal returns from grazing cattle. Regression analysis suggests that urine N2O EFs were controlled more by composition than was the case for dung, whilst dung N2O EFs were more related to soil and environmental factors. The urine N2O EF was significantly greater from the site in SW England, and significantly greater from the early grazing season urine application than later applications. Dycandiamide reduced the N2O EF from urine patches by an average of 46%. The significantly lower excretal EF3 than the IPCC default has implications for the UK's national inventory and for subsequent carbon footprinting of UK ruminant livestock products.

Critical review of the impacts of grazing intensity on soil organic carbon storage and other soil quality indicators in extensively managed grasslands.

Livestock grazing intensity (GI) is thought to have a major impact on soil organic carbon (SOC) storage and soil quality indicators in grassland agroecosystems. To critically investigate this, we conducted a global review and meta-analysis of 83 studies of extensive grazing, covering 164 sites across different countries and climatic zones. Unlike previous published reviews we normalized the SOC and total nitrogen (TN) data to a 30 cm depth to be compatible with IPCC guidelines. We also calculated a normalized GI and divided the data into four main groups depending on the regional climate (dry warm, DW; dry cool, DC; moist warm, MW; moist cool, MC). Our results show that taken across all climatic zones and GIs, grazing (below the carrying capacity of the systems) results in a decrease in SOC storage, although its impact on SOC is climate-dependent. When assessed for different regional climates, all GI levels increased SOC stocks under the MW climate (+7.6%) whilst there were reductions under the MC climate (-19%). Under the DW and DC climates, only the low (+5.8%) and low to medium (+16.1%) grazing intensities, respectively, were associated with increased SOC stocks. High GI significantly increased SOC for C4-dominated grassland compared to C3-dominated grassland and C3-C4 mixed grasslands. It was also associated with significant increases in TN and bulk density but had no effect on soil pH. To protect grassland soils from degradation, we recommend that GI and management practices should be optimized according to climate region and grassland type (C3, C4 or C3-C4 mixed).

Cytoplasmic PML promotes TGF-ß-associated epithelial-mesenchymal transition and invasion in prostate cancer.

Epithelial-mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-ß signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-ß signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-ß canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-ß signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.

Simulation of nitrous oxide emissions at field scale using the SPACSYS model.

Nitrous oxide emitted to the atmosphere via the soil processes of nitrification and denitrification plays an important role in the greenhouse gas balance of the atmosphere and is involved in the destruction of stratospheric ozone. These processes are controlled by biological, physical and chemical factors such as growth and activity of microbes, nitrogen availability, soil temperature and water availability. A comprehensive understanding of these processes embodied in an appropriate model can help develop agricultural mitigation strategies to reduce greenhouse gas emissions, and help with estimating emissions at landscape and regional scales. A detailed module to describe the denitrification and nitrification processes and nitrogenous gas emissions was incorporated into the SPACSYS model to replace an earlier module that used a simplified first-order equation to estimate denitrification and was unable to distinguish the emissions of individual nitrogenous gases. A dataset derived from a Scottish grassland experiment in silage production was used to validate soil moisture in the top 10 cm soil, cut biomass, nitrogen offtake and N2O emissions. The comparison between the simulated and observed data suggested that the new module can provide a good representation of these processes and improve prediction of N2O emissions. The model provides an opportunity to estimate gaseous N emissions under a wide range of management scenarios in agriculture, and synthesises our understanding of the interaction and regulation of the processes.

Assessing the toxic effects of DMSO on cord blood to determine exposure time limits and the optimum concentration for cryopreservation.

BACKGROUND AND OBJECTIVES: Advantages of using cord blood (CB) over other sources of haematopoietic progenitor cells, such as bone marrow, include the ability to cryopreserve and bank the samples until requested for a transplant. Cryopreservation requires the addition of a cryoprotectant to prevent the formation of intracellular ice during freezing. Dimethyl sulphoxide (DMSO) is commonly used at a concentration of 10% (v/v); however, there is evidence to suggest this chemical is toxic to cells as well as to patients after infusion. MATERIALS AND METHODS: The toxic effects of DMSO were assessed through cell viability and in vitro functional assays in fresh and post-thaw CB samples before determining the maximum exposure time and optimal concentration for cryopreservation. RESULTS: A dose-dependent toxicity of DMSO was observed in fresh samples with 40% removing all viable and functional haematopoietic progenitor cells (HPC). In fresh and post-thaw analysis, minimal toxic effect was observed when cryopreservation was delayed for up to 1 h after 10% DMSO addition. After thawing, DMSO washout was superior to dilution or unmanipulated when maintained for long periods (advantage observed 1 h after thawing). Finally, the optimum concentration for cryopreserving CB was found to be 7.5 to 10% with detrimental effects observed outside of this range. CONCLUSION: These results support the use of 7.5-10% as the optimal DMSO concentration and the maximum exposure time should be limited to <1 h prior to freezing and 30 min post-thaw.

Canine tick-borne pathogens and associated risk factors in dogs presenting with and without clinical signs consistent with tick-borne diseases in northern Australia.

OBJECTIVES: To estimate the proportion of canine tick-borne disease (CTBD) pathogens in dogs from northern states of Australia presenting with and without clinical signs/laboratory abnormalities suggestive of CTBD and to evaluate associated risk factors. DESIGN: Client-owned dogs presented to a general practice clinic in the Northern Territory (NT; n = 138) and five referral hospitals in south-east Queensland (SEQ; n = 100) were grouped into CTBD-suspect and -control groups based on clinical and laboratory criteria. Blood and sera were screened for haemotropic Mycoplasma spp., Babesia spp., Anaplasma spp., Ehrlichia spp. and Hepatozoon spp. using microscopic examination, in-clinic ELISA testing and PCR assays. Dog-specific risk factors associated with the presence of CTBD pathogens were evaluated. RESULTS: Overall, 24.4% of the suspect group and 12.2% of the control group dogs were infected. The proportions of M. haemocanis, B. vogeli, A. platys, Candidatus Mycoplasma haematoparvum, and C. Mycoplasma haemobos were 7.1%, 5.0%, 3.8%, 1.7% and 0.4%, respectively. Dogs originating from the NT were 3.6-fold (95% confidence interval (CI) 1.51-8.62; P = 0.004) more likely to be infected with CTBD pathogens than those from SEQ. Male dogs were 2.3-fold (95% CI 1.17-4.80, P = 0.024) more likely to be PCR-positive to CTBD pathogens than female dogs. Dogs presenting with clinical signs consistent with CTBD and thrombocytopenia were more likely to be infected by CTBD pathogens (odds ratio 2.85; 95% CI 1.16, 7.02; P = 0.019). CONCLUSIONS: Haemotropic mycoplasmas were the most common tick-borne pathogen infecting client-owned dogs. Subclinical cases were common in dogs from the NT. Veterinary practitioners should be aware of the proportion of CTBD pathogens and the presenting features of clinical and subclinical disease in their area.

Nitrous oxide emissions from cattle excreta applied to a Scottish grassland: effects of soil and climatic conditions and a nitrification inhibitor.

Dung and urine excreted onto grasslands are a major source of nitrous oxide (N2O). These N2O emissions stem from inefficient utilisation of nitrogen (N) ingested by ruminants, and the inability of pasture to utilise the deposited N. Predicted growth in dairy and meat consumption means that there is a requirement to quantify N2O emissions, and investigate emission reduction mechanisms. Three 12 month 'seasonal' experiments were undertaken at Crichton, SW Scotland, where N2O emissions were measured from applications of cattle urine, dung, artificial urine and urine+a nitrification inhibitor (NI), dicyandiamide (DCD). The three application timings were 'spring', 'summer' and 'autumn', representative of early-, mid- and late grazing seasons. N2O emissions were measured from static chambers for 12 months. The aim was to quantify emissions from cattle excreta, and determine their dependence on the season of application, and the respective contribution of dung and urine to total excreta emissions. Measurement from NI amended urine was made to assess DCD's potential as an emission mitigation tool. Emissions were compared to the IPCC's default emission factor (EF) of 2% for cattle excreted N. Mean annual cumulative emissions from urine were the highest when applied in summer (5034 g N2O-N ha(-1)), with lower emissions from spring (1903 g N2O-N ha(-1)) and autumn (2014 g N2O-N ha(-1)) application, most likely due to higher temperatures and soil moisture conducive to both nitrification and denitrification in the summer months. Calculated EFs were significantly greater from urine (1.1%) than dung (0.2%) when excreta was applied in summer, and EFs varied with season of application, but in all experiments were lower than the IPCC default of 2%. These results support both lowering and disaggregating this EF into individual EFs for dung and urine. Addition of DCD to urine caused no significant reduction in emissions, suggesting that more research is required into its use as a mitigation option.

Susceptibility of cat fleas (Siphonaptera: Pulicidae) to fipronil and imidacloprid using adult and larval bioassays.

The monitoring of the susceptibility offleas to insecticides has typically been conducted by exposing adults on treated surfaces. Other methods such as topical applications of insecticides to adults and larval bioassays on treated rearing media have been developed. Unfortunately, baseline responses of susceptible strains of cat flea, Ctenocephalides felis (Bouchè), except for imidacloprid, have not been determined for all on-animal therapies and new classes of chemistry now being used. However, the relationship between adult and larval bioassays of fleas has not been previously investigated. The adult and larval bioassays of fipronil and imidacloprid were compared for both field-collected isolates and laboratory strains. Adult topical bioassays of fipronil and imidacloprid to laboratory strains and field-collected isolates demonstrated that LD50s of fipronil and imidacloprid ranged from 0.11 to 0.40 nanograms per flea and 0.02 to 0.18 nanograms per flea, respectively. Resistance ratios for fipronil and imidacloprid ranged from 0.11 to 2.21. Based on the larval bioassay published for imidacloprid, a larval bioassay was established for fipronil and reported in this article. The ranges of the LC50s of fipronil and imidacloprid in the larval rearing media were 0.07-0.16 and 0.11-0.21 ppm, respectively. Resistance ratios for adult and larval bioassays ranged from 0.11 to 2.2 and 0.58 to 1.75, respectively. Both adult and larval bioassays provided similar patterns for fipronil and imidacloprid. Although the adult bioassays permitted a more precise dosage applied, the larval bioassays allowed for testing isolates without the need to maintain on synthetic or natural hosts.

HAGE (DDX43) is a biomarker for poor prognosis and a predictor of chemotherapy response in breast cancer.

BACKGROUND: HAGE protein is a known immunogenic cancer-specific antigen. METHODS: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. RESULTS: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGE+) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGE+expression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+residual disease (P=0.0003). CONCLUSIONS: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC.

The helicase HAGE prevents interferon-α-induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1.

The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5+ malignant melanoma-initiating cells (ABCB5+ MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK-STAT (janus kinase-signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK-STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro. Finally, using a stem cell proliferation assay and tumour xenotransplantation assay in non-obese diabetic/severe combined immunodeficiency mice, we show that HAGE promotes MMICs-dependent tumour initiation and tumour growth by preventing the anti-proliferative effects of interferon-α (IFNα). Our results suggest that the helicase HAGE has a key role in the resistance of ABCB5+ MMICs to IFNα treatment and that cancer therapies targeting HAGE may have broad implications for the treatment of malignant melanoma.

Potential of legume-based grassland-livestock systems in Europe: a review.

European grassland-based livestock production systems face the challenge of producing more meat and milk to meet increasing world demands and to achieve this using fewer resources. Legumes offer great potential for achieving these objectives. They have numerous features that can act together at different stages in the soil-plant-animal-atmosphere system, and these are most effective in mixed swards with a legume proportion of 30-50%. The resulting benefits include reduced dependence on fossil energy and industrial N-fertilizer, lower quantities of harmful emissions to the environment (greenhouse gases and nitrate), lower production costs, higher productivity and increased protein self-sufficiency. Some legume species offer opportunities for improving animal health with less medication, due to the presence of bioactive secondary metabolites. In addition, legumes may offer an adaptation option to rising atmospheric CO2 concentrations and climate change. Legumes generate these benefits at the level of the managed land-area unit and also at the level of the final product unit. However, legumes suffer from some limitations, and suggestions are made for future research to exploit more fully the opportunities that legumes can offer. In conclusion, the development of legume-based grassland-livestock systems undoubtedly constitutes one of the pillars for more sustainable and competitive ruminant production systems, and it can be expected that forage legumes will become more important in the future.

Virulence loss and amastigote transformation failure determine host cell responses to Leishmania mexicana.

The effect of alterations in virulence and transformation by long-term in vitro culture of Leishmania mexicana promastigotes on infectivity and immune responses was investigated. Fresh parasite cultures harvested from Balb/c mice were passaged 20 times in vitro. Infectivity was decreased and was completely avirulent after 20 passages. The qPCR results showed a down-regulation of GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3 genes after passage seven concomitant with a reduced and absence of infectivity by passages seven and 20, respectively. Parasites at passages one and 20 are referred to as virulent and avirulent, respectively. The growth of avirulent and virulent parasite was affected by conditioned media derived from macrophages or monocytes infected with parasites for 2 h. Giemsa staining showed the failure of avirulent but not virulent parasites to transform to the amastigote stage in infected host cells with both virulent and avirulent modulating the expression of CCL-22, Tgad51, Cox2, IL-1, IL-10, TGF-ß, TNF-α, Rab7, Rab9 and A2 genes; virulent but not avirulent L. mexicana significantly up-regulated Th2-associated cytokines, but down-regulated Rab7 and Rab9 gene expression. In conclusion, a model for L. mexicana is reported, which is of potential value in studying host-parasite interaction.

Expression of transglutaminase-2 isoforms in normal human tissues and cancer cell lines: dysregulation of alternative splicing in cancer.

The multiple enzymatic activities and functions of transglutaminase type 2 (TG2) may be attributed to alternative TG2 molecules produced by differential splicing of TG2 mRNA. Different RNA transcripts of the human TG2 gene (TGM2) have been identified, but the expression of TG2 multiple transcripts has never been systematically addressed. We have confirmed and rationalized the main TG2 variants and developed a screening assay for the detection of alternative splicing of TG2, based on real-time reverse-transcription PCR. We have quantified the multiple TG2 transcripts in a wide range of normal tissues and in cancer cell lines from four different sites of origin. Our data show a significant correlation in the expression of canonical and alternative TG2 isoforms in normal human tissue, but differences in alternative splicing of TG2 in cancer cell lines, suggesting that in cancer cells the alternative splicing of TG2 is a more active process.

Canine vector-borne disease pathogens in dogs from south-east Queensland and north-east Northern Territory.

OBJECTIVES: To determine the prevalence of canine vector-borne diseases (CVBD: Babesia spp., Anaplasma spp., Ehrlichia spp., haemotropic mycoplasmas and Hepatozoon) in Australian dogs; namely, dogs from pounds in south-east Queensland and an indigenous Aboriginal community in the north-east of the Northern Territory. DESIGN AND PROCEDURE: Blood samples were collected from 100 pound dogs and 130 Aboriginal community dogs and screened for the CVBD pathogens using polymerase chain reaction (PCR). All positive PCR products were sequenced for species confirmation. RESULTS: In total, 3 pound dogs and 64 Aboriginal community dogs were infected with at least one CVBD pathogen. Overall, B. vogeli was detected in 13 dogs, A. platys in 49, M. haemocanis in 23, Candidatus Mycoplasma haematoparvum in 3 and C. M. haemobos in 1 dog. Co-infections were detected in 22 Aboriginal community dogs. CONCLUSIONS: This study found B. vogeli, A. platys and haemotropic mycoplasma infections to be common in dogs in subtropical and tropical areas of Australia. This study also reports for the first time the prevalence and genetic characterisation of haemotropic mycoplasmas in dogs in Australia.

UK emissions of the greenhouse gas nitrous oxide.

Signatories of the Kyoto Protocol are obliged to submit annual accounts of their anthropogenic greenhouse gas emissions, which include nitrous oxide (N(2)O). Emissions from the sectors industry (3.8 Gg), energy (14.4 Gg), agriculture (86.8 Gg), wastewater (4.4 Gg), land use, land-use change and forestry (2.1 Gg) can be calculated by multiplying activity data (i.e. amount of fertilizer applied, animal numbers) with simple emission factors (Tier 1 approach), which are generally applied across wide geographical regions. The agricultural sector is the largest anthropogenic source of N(2)O in many countries and responsible for 75 per cent of UK N(2)O emissions. Microbial N(2)O production in nitrogen-fertilized soils (27.6 Gg), nitrogen-enriched waters (24.2 Gg) and manure storage systems (6.4 Gg) dominate agricultural emission budgets. For the agricultural sector, the Tier 1 emission factor approach is too simplistic to reflect local variations in climate, ecosystems and management, and is unable to take into account some of the mitigation strategies applied. This paper reviews deviations of observed emissions from those calculated using the simple emission factor approach for all anthropogenic sectors, briefly discusses the need to adopt specific emission factors that reflect regional variability in climate, soil type and management, and explains how bottom-up emission inventories can be verified by top-down modelling.

Immunogenicity of MHC Class I Peptides Derived from Leishmania mexicana Gp63 in HLA-A2.1 Transgenic (HHDII) and BALB/C Mouse Models.

BACKGROUND: Leishmania is an intracellular parasite infecting humans and many wild and domestic animals. Recent studies have suggested an important role for cytotoxic T cells against Leishmania. Peptide-based vaccines targeting short sequences derived from known immunogenic proteins have been shown to elicit cellular immune responses against disparate pathogens. METHODS: We predicted four HLA-A2 peptides derived from L. mexican/major gp63 and tested these in HHD II mice, as well as four peptides for mouse MHC class I from the same proteins tested in BALB/ mice. RESULTS: The results revealed immunogenicity for three of the four peptides predicted for HLA-A2. Immunisation with these peptides, along with IFA, induced CTL responses detected by standard 4-hour cytotoxicity assay and significantly upregulated the production of IFN-γ. When HHDII mice were injected IM with L. mexicana gp63 cDNA and splenocytes were restimulated with blasts loaded with the immunogenic peptides, two of the peptides were able to induce significant level of IFN-γ detected by ELISA. None of the peptides predicted for Balb/c mouse MHC class I elicited CTL activity or significantly upregulated the IFN-γ. CONCLUSION: The results may help in developing a peptide-based vaccine, which can be applied alone or in combination with drugs against Leishmania.