Evaluation of a biodegradable microparticulate polymer as a carrier for Burkholderia pseudomallei subunit vaccines in a mouse model of melioidosis.

Melioidosis, a potentially lethal disease of humans and animals, is caused by the soil-dwelling bacterium Burkholderia pseudomallei. Due to B. pseudomallei's classification as a Tier 1 Select Agent, there is substantial interest in the development of an effective vaccine. Yet, despite decades of research, no effective target, adjuvant or delivery vehicle capable of inducing protective immunity against B. pseudomallei infection has been identified. We propose a microparticulate delivery vehicle comprised of the novel polymer acetalated dextran (Ac-DEX). Ac-DEX is an acid-sensitive biodegradable carrier that can be fabricated into microparticles (MPs) that are relatively stable at pH 7.4, but rapidly degrade after phagocytosis by antigen presenting cells where the pH can drop to 5.0. As compared to other biomaterials, this acid sensitivity has been shown to enhance cross presentation of subunit antigens. To evaluate this platform as a delivery system for a melioidosis vaccine, BALB/c mice were vaccinated with Ac-DEX MPs separately encapsulating B. pseudomallei whole cell lysate and the toll-like receptor (TLR) 7/8 agonist resiquimod. This vaccine elicited a robust antibody response that included both Th1 and Th2 immunity. Following lethal intraperitoneal challenge with B. pseudomallei 1026b, vaccinated mice demonstrated a significant delay to time of death compared to untreated mice. The formulation, however, demonstrated incomplete protection indicating that lysate protein offers limited value as an antigen. Nevertheless, our Ac-DEX MPs may offer an effective delivery vehicle for a subunit B. psuedomallei vaccine.

Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile.

DNA microarrays and two-dimensional (2-D) gel electrophoresis were utilized to analyze the global effect of bile on transcription and protein synthesis in Salmonella enterica serovar Typhimurium. Two bile-regulated proteins, YciF and PagC, were identified by 2-D gel electrophoresis and mass spectrometry fingerprinting. The operon yciGFE-katN demonstrated increased transcriptional activity in the presence of bile. While this operon has previously been shown to be RpoS-regulated, data from this study suggested that yciGFE-katN is regulated by bile independent of RpoS. The PhoP-PhoQ-regulated PagC is decreased in the presence of bile. Characterization of the untranslated leader of pagC demonstrated that a 97-bp region is necessary for the bile-mediated repression of this promoter. Analysis of data from the DNA microarray revealed an effect of bile on important global mechanistic pathways in S. enterica serovar Typhimurium. Genes involved in type III secretion-mediated invasion of epithelial cells demonstrated an overall repression of transcription in the presence of bile, corroborating previously reported data from this laboratory [Infect. Immun. 68 (2000) 6763]. In addition, bile-mediated transcriptional repression of genes involved in flagellar biosynthesis and motility was observed. These data further demonstrate that bile is an important environmental signal sensed by Salmonella spp. and that bile plays a role in regulating bacterial gene expression in multiple virulence-associated pathways.

Do we really understand? An experiential exercise for training family therapists.

This paper presents an experiential exercise for training beginning marriage and family therapists, as well as several adaptations of the format. The basic exercise consists of role playing a therapy session in which the people playing the clients speak a language that is not understood by the therapist-in-training. Although the therapist cannot understand the clients, the clients can understand the therapist. This exercise assists students to become more cognizant of the need to be cautious and curious in the cocreation of meaning in therapy. In addition, this exercise builds confidence and clinical skills, such as learning about nonverbal communication while observing process.

Change, disengagement, and renewal: relationship dynamics between young adults and their fathers after divorce.

Little is known about children's perceptions of their parents' divorce or how children construct meaning around the divorce and their subsequent relationships with their parents. The focus of this study was to learn about the experiences and the meanings young adults had constructed about the divorce process and their relationships with their fathers in the years after the divorce. The findings revealed a broad spectrum of experiences and several key issues that gave meaning to both the disengagement and the reengagement with their fathers. Loss, trust, acceptance, availability, and support are a few of the vital issues addressed. Implications for family therapists are discussed.

Methods of feminist family therapy supervision.

Although feminist family therapy has been studied and practiced for more than 20 years, writing about feminist supervision in family therapy has been limited. Three supervision methods emerged from a qualitative study of the experiences of feminist family therapy supervisors and the therapists they supervised: The supervision contract, collaborative methods, and hierarchical methods. In addition to a description of the participants' experiences of these methods, we discuss their fit with previous theoretical descriptions of feminist supervision and offer suggestions for future research.

Two measures of preference during forced-choice assessments.

A forced-choice preference assessment was conducted in which two dependent measures were used to select preferred stimuli: (a) approach responding and (b) latency to the first aberrant response. Stimuli identified as preferred based on both dependent measures were then evaluated during treatment. The results suggested that latency may be a useful measure in the selection of preferred stimuli during forced-choice assessments.

Comparative analysis of Salmonella enterica serovar Typhimurium biofilm formation on gallstones and on glass.

In this study, the roles of global regulators, motility, lipopolysaccharide, and exopolysaccharides were further characterized with respect to biofilm formation on both gallstones and glass surfaces. These studies show the complex nature of biofilms and demonstrate that characteristics observed for each biofilm are unique to the particular culture condition.

Salmonella enterica serovar typhimurium invasion is repressed in the presence of bile.

As enteric pathogens, the salmonellae have developed systems by which they can sense and adapt appropriately to deleterious intestinal components that include bile. Previously, growth in the presence of bile was shown to repress the transcription of prgH, a locus encoding components of the Salmonella pathogenicity island I (SPI-1) type III secretion system (TTSS) necessary for eukaryotic cell invasion. This result suggested an existing interaction between salmonellae, bile, and eukaryotic cell invasion. Transcription assays demonstrated that invasion gene regulators (e.g., sirC and invF) are repressed by bile. However, bile does not interact with any of the invasion regulators directly but exerts its effect at or upstream of the two-component system at the apex of the invasion cascade, SirA-BarA. As suggested by the repression of invasion gene transcription in the presence of bile, Western blot analysis demonstrated that proteins secreted by the SPI-1 TTSS were markedly reduced in the presence of bile. Furthermore, Salmonella enterica serovar Typhimurium grown in the presence of bile was able to invade epithelial cells at only 4% of the level of serovar Typhimurium grown without bile. From these data, we propose a model whereby serovar Typhimurium uses bile as an environmental signal to repress its invasive capacity in the lumen of the intestine, but upon mucous layer penetration and association with intestinal epithelial cells, where the apparent bile concentration would be reduced, the system would become derepressed and invasion would be initiated.

Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp.

Salmonellae can exist in an asymptomatic carrier state in the human gallbladder. Individuals with gallstones are more likely to become typhoid carriers, and antibiotic treatments are often ineffectual against Salmonella enterica serovar Typhi in carriers with gallstones. Therefore, we hypothesized that Salmonella spp. form biofilms on the surfaces of gallstones, where the bacteria are protected from high concentrations of bile and antibiotics. A number of methods were utilized to examine biofilm formation on human gallstones and glass coverslips in vitro, including confocal, light, and scanning electron microscopy. In our assays, salmonellae formed full biofilms on the surfaces of gallstones within 14 days and appeared to excrete an exopolysaccharide layer that bound them to the surfaces and to other bacteria. Efficient biofilm formation on gallstones was dependent upon the presence of bile, as a biofilm did not form on gallstones within 14 days in Luria-Bertani broth alone. The biofilms formed by a Salmonella enterica serovar Typhi Vi antigen mutant, as well as strains with mutations in genes that eliminate production of four different fimbriae, were indistinguishable from the biofilms formed by the parents. Mutants with an incomplete O-antigen, mutants that were nonmotile, and mutants deficient in quorum sensing were unable to develop complete biofilms. In addition, there appeared to be selectivity in salmonella binding to the gallstone surface that did not depend on the topology or surface architecture. These studies should aid in the understanding of the Salmonella carrier state, an important but underresearched area of typhoid fever pathogenesis. If the basis of carrier development can be understood, it may be possible to identify effective strategies to prevent or treat this chronic infection.

Bile-salt-mediated induction of antimicrobial and bile resistance in Salmonella typhimurium.

By DNA microarray, the Salmonella typhimurium marRAB operon was identified as being bile-activated. Transcriptional assays confirm that marRAB is activated in the presence of bile and that this response is concentration-dependent. The bile salt deoxycholate is alone able to activate transcription, while there was no response in the presence of other bile salts tested or a non-ionic detergent. Deoxycholate is able to interact with MarR and interfere with its ability to bind to the mar operator. In addition, incubation of salmonellae in the presence of sublethal concentrations of bile is able to enhance resistance to chloramphenicol and bile, by means of both mar-dependent and mar-independent pathways. To further characterize putative marRAB-regulated genes that may be important for the resistance phenotype, acrAB, which encodes an efflux pump, was analysed. In S. typhimurium, acrAB is required for bile resistance, but while transcription of acrAB is activated by bile, this activation is independent of marRAB, as well as Rob, RpoS or PhoP-PhoQ. These data suggest that bile interacts with salmonellae to increase resistance to bile and other antimicrobials and that this can occur by marRAB- and acrAB-dependent pathways that function independently with respect to bile activation.