A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.SMGT ͉ hyperacute rejection ͉ swine

Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods to make them sufficiently effective in the clinic. Similarly, it is clear that additional strategies are required to activate effective immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by malignant cells to prevent immune cell function. Combined or single strategies to enhance the efficacy and immunogenicity of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated. Some representative DNA cancer vaccine studies are also discussed.

The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8+ T cells, and NK cells, into the tumor microenvironment and are being explored as promising adjuvants in cancer immunotherapies. In this minireview, we discuss how cGAS–STING and RIG-I–MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways are currently being targeted for clinical application in oncology.

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The objective of the present study was to evaluate the diagnostic performance of a noninvasive assay, conjunctival swab (CS) nested-PCR (n-PCR), for diagnosing canine leishmaniasis (CanL) in different stages of infection in comparison to the performance of the indirect immunofluorescence antibody test (IFAT), lymph node microscopy, and buffy coat n-PCR. To this end, we performed a cross-sectional survey among 253 nonselected dogs in areas of endemicity in central Italy. We also performed a longitudinal study of CS n-PCR among 20 sick dogs undergoing antileishmanial treatment. In the first study, among the 72 animals that were positive by at least one test (28.45%), CS n-PCR showed the best relative performance (76.38%), with a high concordance in comparison to standard IFAT serology ( ‫؍‬ 0.75). The highest positivity rates using CS n-PCR were found in asymptomatic infected dogs (84.2%) and sick dogs (77.8%); however, the sensitivity of the assay was not associated with the presence of clinical signs. In the follow-up study on treated sick dogs, CS n-PCR was the most sensitive assay, with promising prognostic value for relapses. The univariate analysis of risk factors for CanL based on CS n-PCR findings showed a significant correlation with age (P ‫؍‬ 0.012), breed size (P ‫؍‬ 0.026), habitat (P ‫؍‬ 4.9 ؋ 10 ؊4 ), and previous therapy (P ‫؍‬ 0.014). Overall, the results indicated that CS n-PCR was the most sensitive assay of the less invasive diagnostic methods and could represent a good option for the early and simple diagnosis of CanL infection in asymptomatic animals and for monitoring relapses in drug-treated dogs.

Cryptosporidium and Giardia are two of the most common enteric pathogens of domestic and wild animals and humans. However, little is known on the prevalence, clinical manifestations and economic and zoonotic significance of these infections in horses. This study was undertaken to investigate the prevalence, excretion patterns and risk factors related to the faecal shedding of Cryptosporidium oocysts and Giardia cysts in horses and the zoonotic potential of species/genotypes isolated. The survey was performed on 120 foals and 30 broodmares reared in five Italian farms. Foals were divided in four homogeneous groups of 30 animals each (age classes: 0-2, 2-4, 4-8, >8 weeks). Three sequential faecal samples were collected from each animal and analysed by three techniques: direct fluorescent antibody test (DFA), faecal flotation (FF) and stained faecal smears (SFS). The DFA results showed a prevalence of 8% for Cryptosporidium and of 13.33% for Giardia; the prevalence values obtained by FF and SFS were lower and in poor agreement with DFA results. Giardia and Cryptosporidium infections were more common in foals (23.33% and 26.66% respectively) and higher excretions were observed in the youngest foals. Distribution of Cryptosporidium prevalence was statistically related to farms (P < 0.01), age of animals (P < 0.01), but was unrelated to the presence of diarrhoea. In the case of Giardia, the prevalence was only related to age (P < 0.01). Pattern sheddings were related to intestinal diseases and horse age (P < 0.01). Risk factors for shedding included residence farms and age older than 8 weeks for both parasites. All DFA-positive faecal samples were submitted to DNA extraction and PCR to determine Giardia and Cryptosporidium species/genotypes. Sequence analysis of the COWP gene of Cryptosporidium and of the SSU-rRNA gene of Giardia revealed that they were identical to each other and identified Cryptosporidium parvum and Giardia duodenalis assemblage E. The potential role of infected horses in zoonotic transmission of Cryptosporidium was supported by the findings of this study.

ABSTRACT. In order to investigate the prevalence of tick-borne diseases, equine piroplasmosis, equine granulocytic anaplasmosis and Lyme borreliosis in Central Italy, blood samples from 300 horses were analyzed for the presence of antibodies against Babesia caballi, Theileria equi, Anaplasma phagocytophilum and Borrelia burgdorferi using the IFAT. The blood samples were also subjected to PCR assays in order to detect pathogen DNA. A total of 78 (26.0%) and 123 (41.0%) horses were found to be seropositive for B. caballi and T. equi, respectively, while 41 (13. 4%) and 21 (7.0%) horses were, respectively, seropositive for A. phagocytophilum and B. burgdorferi. Seropositivity for more than one agent was detected in 76 horses using IFAT. The most common association observed was between T. equi and B. caballi (14.7%). In addition, 54 horses (18.0%) were found to be positive for one or more tick-borne pathogens (TBPs) using PCR testing. Among these, 28 (9.3%) harbored single infections, while 26 (8.7%) were found to be co-infected with two or more pathogens. The correlation (K value) between IFAT and PCR results was 0.32 for T. equi, 0.34 for B. caballi, 0.62 for B. burgdorferi and 0.48 for A. phagocytophilum, reflecting an unprecedented degree of multiple exposures to TBPs in horses.

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