Aims: To evaluate the suitability of using sterile water and phosphate-buffered saline (PBS) for preservation of bacteria pathogenic to plants or humans. Methods and Results: The stationary-phase bacterial cells collected from rich agar media were transferred to 10 ml of sterile water or PBS (pH 7AE2) containing KH 2 PO 4 , 15AE44 lM M; NaCl, 1AE55 mM M; Na 2 HPO 4 , 27AE09 lM M in a screw-cap tube. The tubes were sealed with parafilm membranes and stored in the dark and at room temperature. Almost all the bacteria tested (148 strains), including Pseudomonas fluorescens, P. viridiflava, Erwinia spp., Xanthomonas campestris, Cytophaga johnsonae, Salmonella spp., Yersinia enterocolitica, Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus, survived in water for at least several months and up to 16 years. A vast majority of the Gram-negative bacteria tested survived equally well in water and in PBS for at least 30 weeks. However, the populations of two Gram-positive bacteria [G(+)], L. monocytogenes and Staph. aureus, declined more rapidly in water than in PBS. Conclusions: Plant-and human-pathogenic bacteria can be preserved in pure water or PBS for several years. G(+) bacteria appear to survive better in PBS than in water. Significance and Impact of the Study: The method described here is a simple and economical means for preservation of bacterial cultures, which is especially useful for laboratories not equipped with the lyophilizer or ultra-low freezer. Long-term survival of food-borne pathogens in water underlines the importance of water as a potential vehicle for transmitting the diseases.

This study clarifies the mechanism by which naked DNA is eliminated from the circulation after i.v. injection, focusing on the role of both the liver and blood compartments in vivo (i.e. mouse). With this knowledge, we can more clearly understand the mechanism of naked DNA clearance and develop more efficient strategies for DNA transfer in vivo.

The vectors for gene delivery are usually classified as viral and nonviral vectors. While the viral vectors are very efficient in transducing cells, safety concerns regarding their use in humans make nonviral vectors an attractive alternative. Among the nonviral vectors, the lipoplexes (complexes of cationic liposome/pDNA) are the most studied and represent the most promising approaches for human clinical trials. However, an inflammatory response is invariably associated with administration of the lipoplexes, which must be avoided in the clinical application. Here, we have successfully developed a nonimmunostimulatory vector for gene therapy. The vector possesses dual functions of: 1) efficiently delivering a gene to target cells and 2) codelivering DNA and inflammatory suppressors into the immune cells where the released suppressor can inhibit cytokine production. The inflammatory suppressors successfully delivered by the vector included glucocorticoids, a nonsteroidal anti-inflammatory drug (NSAID), an NF-kappaB inhibitor, and a natural compound from an herbal medicine. Intravenous injection of the vector dramatically suppressed the cytokine production induced by CpG motif pDNA, including TNF-alpha, IL-12 and IFN-gamma. This new gene vector has a great potential in clinical gene therapy. Another potential use of the vector is codelivery of an enhancer candidate, acting at the transcriptional and translational levels to improve the efficiency of gene transfer by the nonviral vector. Moreover, the unique feature of this vector is that it can be used as an easy and powerful tool for in vivo screening of anti-inflammatory drugs.

This study provides new insights into the mechanism of electro-gene transfer, and may allow the definition of newer and more efficient conditions for in vivo electroporation.

bVaccination remains the most effective public health tool to prevent infectious diseases. Many vaccines are marginally effective and need enhancement for immunocompromised, elderly, and very young populations. To enhance immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create VacSIM (vaccine self-assembling immune matrix), a new delivery method. VacSIM solution can easily be mixed with antigens, organisms, and adjuvants for injection. Postinjection, the peptides self-assemble into hydrated nanofiber gel matrices, forming a depot with antigens and adjuvants in the aqueous phase. We believe the depot provides slow release of immunogens, leading to increased activation of antigen-presenting cells that then drive enhanced immunogenicity. Using recombinant hepatitis B virus surface antigen (rHBsAg) as a model immunogen, we compared VacSIM delivery to delivery in alum or complete Freund's adjuvant (CFA). Delivery of the rHBsAg antigen to mice via VacSIM without adjuvant elicited higher specific IgG responses than when rHBsAg was delivered in alum or CFA. Evaluating IgG subtypes showed a mixed Th1/Th2 type response following immunization with VacSIM, which was driven further toward Th1 with addition of CpG as the adjuvant. Increased specific IgG endpoint titers were observed in both C57BL/6 and BALB/c mice, representative of Th1 and Th2 environments, respectively. Restimulation of splenocytes suggests that VacSIM does not cause an immediate proinflammatory response in the host. Overall, these results suggest that VacSIM, as a new delivery method, has the potential to enhance immunogenicity and efficacy of numerous vaccines. Vaccines remain the single greatest public health tool to combat infectious diseases. Vaccine formulation and delivery are key to the ability of vaccines to induce the desired immune responses. One goal of vaccine delivery is to present vaccine antigens in a manner that enhances antigen-presenting-cell (APC) activation, leading to antigen/organism uptake and processing of vaccine antigen(s). Delivery methods or adjuvants that safely enhance vaccine immunogenicity/efficacy are desirable for vaccines that are marginally effective and for vaccines administered to low responders or immunocompromised populations. Additional goals are to reduce the number of doses required to induce effective, vaccine responses and to reduce the amount of vaccine/dose, especially when a single dose of vaccine is administered, as with annual influenza vaccines. Lastly, in pandemics, a vaccine that produces high titers after a single administration would be beneficial. Recent advances in the understanding of how innate mechanisms influence adaptive immunity have led to more rational design in the development of new vaccine adjuvants and delivery systems.Aluminum salts were the first adjuvants licensed for human vaccines in the 1920s. The licensure of non-aluminum salt adjuvants took an additional 80 years (1). One reason for this long gap is that the principles of adjuvant activit...

Despite significant progress, China faces the challenge of re-emerging schistosomiasis transmission in currently controlled areas due, in part, to the presence of a range of animal reservoirs, notably water buffalo and cattle, which can harbor Schistosoma japonicum infections. Environmental, ecological and social-demographic changes in China, shown to affect the distribution of oncomelanid snails, can also impact future schistosomiasis transmission. In light of their importance in the S. japonicum , lifecycle, vaccination has been proposed as a means to reduce the excretion of egg from cattle and buffalo, thereby interrupting transmission from these reservoir hosts to snails. A DNA-based vaccine (SjCTPI) our team developed showed encouraging efficacy against S. japonicum in Chinese water buffaloes. Here we report the results of a double-blind cluster randomized trial aimed at determining the impact of a combination of the SjCTPI bovine vaccine (given as a prime-boost regimen), human mass chemotherapy and snail control on the transmission of S. japonicum in 12 selected administrative villages around the Dongting Lake in Hunan province. The trial confirmed human praziquantel treatment is an effective intervention at the population level. Further, mollusciciding had an indirect ~50% efficacy in reducing human infection rates. Serology showed that the SjCTPI vaccine produced an effective antibody response in vaccinated bovines, resulting in a negative correlation with bovine egg counts observed at all post-vaccination time points. Despite these encouraging outcomes, the effect of the vaccine in preventing human infection was inconclusive. This was likely due to activities undertaken by the China National Schistosomiasis Control Program, notably the treatment, sacrifice or removal of bovines from trial villages, over which we had no control; as a result, the trial design was compromised, reducing power and contaminating outcome measures. This highlights the difficulties in undertaking field trials of this nature and magnitude, particularly over a long period, and emphasizes the importance of mathematical modeling in predicting the potential impact of control intervention measures. A transmission blocking vaccine targeting bovines for the prevention of S. japonicum with the required protective efficacy would be invaluable in tandem with other preventive intervention measures if the goal of eliminating schistosomiasis from China is to become a reality.

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Background Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands. Methods This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11–15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17–29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields. Results The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17–29 days post-blood meal) for either parasite strain. Conclusions Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.