Optimization and qualification of an assay that demonstrates that a FimH vaccine induces functional antibody responses in women with histories of urinary tract infections.

Recurrent urinary tract infections (rUTI) are a serious disease associated with morbidities and mortality. Resistance to the standard of care antibiotics is now widespread because of the continued use of antibiotics among people who suffer from rUTI. We are therefore developing a vaccine to prevent recurrences among patients with rUTI. The antigen of the vaccine is FimH, a bacterial adhesin protein, and the vaccine is adjuvanted with a TLR-4 agonist. In a Phase 1 clinical study evaluating the vaccine, immunized individuals produced FimH-binding antibodies. Here we describe the optimization, qualification, and use of an assay to assess the functionality of these anti-FimH antibodies. The suitability of the assay for its intended purpose was demonstrated by selectivity, specificity, sensitivity, and intra-assay and inter-assay precision. The acceptance criteria were achieved for all parameters including intra-assay precision with ≤10% relative standard deviations and inter-assay precision with ≤25% relative standard deviations. The results presented herein suggest this functional assay will be important for supporting the vaccine's efficacy in future human studies. Furthermore and of great significance, these results prove that vaccine-induced functional antibodies can be elicited in rUTI patients against an essential virulence factor, FimH.

Safety and immunogenicity of an adjuvanted Escherichia coli adhesin vaccine in healthy women with and without histories of recurrent urinary tract infections: results from a first-in-human phase 1 study.

Antibiotic resistance among gram-negative bacteria continues to rise globally at an alarming rate. New vaccines that prevent bacterial infections and reduce antibiotic use could provide a potential solution to these problems. This study focused on development of an investigational vaccine to prevent recurrent urinary traction infections (UTI) caused by gram-negative bacteria that use type 1 pili to adhere to, invade, and colonize human bladders. The vaccine antigen is FimH, an adhesin protein on the tip of type 1 pili with a lectin binding domain that enables attachment to glycoproteins on mammalian bladders. This was a phase 1, open-label, dose escalation study evaluating the vaccine in 67 healthy women with and without histories of recurrent UTI. The objectives of the study were to evaluate the safety, tolerability, and immunogenicity of different dosages of the antigen and adjuvant of the vaccine. All dosages were well-tolerated and a low incidence of systemic reactions occurred. No serious adverse events related to the vaccine were reported. The vaccine induced both binding and functional antibodies. The women with histories of recurrent UTI demonstrated greater than 150-fold increases in antibodies against the N-terminal region of FimH. Based on the results of this phase 1 study, this vaccine is proceeding to a double-blind, randomized, placebo-controlled phase 2 study. If this vaccine is successful in future studies, it could potentially prevent millions of recurrent UTI globally and reduce the development of antibiotic resistance.

Bifidenone: Structure-Activity Relationship and Advanced Preclinical Candidate.

Bifidenone is a novel natural tubulin polymerization inhibitor that exhibits antiproliferative activity against a range of human cancer cell lines, making it an attractive candidate for development. A synthetic route was previously developed to alleviate supply constraints arising from its isolation in microgram quantities from a Gabonese tree. Using that previously published route, we present here 42 analogues that were synthesized to examine the structure-activity relationship of bifidenone derivatives. In addition to in vitro cytotoxicity data, data from murine xenograft and pharmacokinetic studies were used to evaluate the analogues. Compounds 45b and 46b were found to demonstrate promising efficacy in murine xenograft experiments, and 46b had significantly more potent in vitro antiproliferative activity against taxane-resistant cell lines compared to that of paclitaxel.

Isolation and Identification of the Novel Tubulin Polymerization Inhibitor Bifidenone.

The pursuit of structurally novel compounds has led to the isolation of a series of neolignans (2-6), for which the structures have been determined from microgram quantities using microcryoprobe NMR technology. Compounds 2-6 provided some unexpectedly clear structure-activity relationship data, with compound 2 demonstrating significantly more potency in the in vitro cytotoxicity assay than the other analogues. Further screening found that compound 2 induces apoptosis with activation of caspase 3/7. The NCI Compare algorithm suggested that compound 2 acts through the inhibition of tubulin/microtubule dynamics. Compound 2 was confirmed to be a tubulin polymerization inhibitor that binds directly to tubulin.

Maintenance of host leukocytes in peripheral immune compartments following lethal irradiation and bone marrow reconstitution: implications for graft versus host disease.

Bone marrow reconstitution is utilized as a tool for disease treatment and as a research technique to elucidate the function of bone marrow derived cells. Clinically successful engraftment is indicated by the development of a functioning immune repertoire. In research, reconstitution is considered successful if >85% of splenic leukocytes are of donor origins. Previous work suggests that splenic reconstitution may not be indicative of reconstitution in the mucosa. We sought to evaluate mucosal reconstitution in animals following a standard bone marrow eradication and reconstitution technique. Bone marrow was harvested from adult B6.SJL donor mice (CD45.1) and injected via either the retro-orbital or intraperitoneal route into lethally irradiated B6 (CD45.2) adult or neonatal recipients respectively. The expression of CD45 by flow cytometry was used to calculate reconstitution with respect to immune compartment and cell type. In reconstituted adult animals 93.2±1.5% of splenic leukocytes expressed the donor CD45.1 antigen thus meeting the standard definition of reconstitution, however only 58.6±13.6% of intestinal lamina propria lymphocytes and 52.4±16.0% of intestinal intraepithelial lymphocytes were of donor origin, confirming splenic reconstitution fails to represent peripheral immune reconstitution. T-cells in the gastrointestinal tract are the most poorly reconstituted, while B-cells appear to be almost universally replaced by donor cells. The inadequate mucosal reconstitution was not corrected by evaluating later time points or by performing the bone marrow transfer during the neonatal period. This demonstration that substantial host T-cells remain in the intestinal mucosa after a "successful" bone marrow transplantation should cause a re-evaluation of data from research bone marrow chimera experiments, as well as the mechanisms for complications after clinical bone marrow transplantation.

Monitoring real-time enzymatic hydrolysis of Distillers Dried Grains with Solubles (DDGS) by dielectric spectroscopy following hydrothermal pre-treatment by steam explosion.

Dielectric spectroscopy (DS) has been used to monitor the simultaneous saccharification and fermentation of lignocellulosic biomass by measuring its dielectric state. However, it is unknown whether following steam explosion (SE) pre-treatment, lignocellulose would still maintain a dielectric state, and, if maintained, whether the dissipation during enzymatic hydrolysis could be monitored. Distillers Dried Grains with Solubles (DDGS), pre-treated by SE, was found to have a capacitance (C = 580 kHz) of approximately 24 pF cm(-1). Following addition of full-strength cellulolytic cocktail A (CC-A; R(2) = 0.97) and 1/3 strength cocktail B (CC-B; R(2) = 0.96), a natural logarithmic decay in capacitance was determined. Furthermore, the DS biomass probes quantified the initial linear rate of dissipation in capacitance during hydrolysis. The rate of CC-B was 34% that of CC-A. These data extend scope and utility of DS biomass probes for monitoring the enzymatic hydrolysis of SE-pre-treated lignocellulosic substrates in real-time.

Cytotoxic and antibacterial beilschmiedic acids from a Gabonese species of Beilschmiedia.

High-throughput natural products chemistry methods have facilitated the isolation of eight new (1-8) and two known (9 and 10) beilschmiedic acid derivatives from the leaves of a Gabonese species of Beilschmiedia. Compounds 3-10 were isolated in microgram quantities, and the NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7 mm MicroCryoProbe. All of the compounds were screened for cytotoxic and antibacterial activity against NCI-H460 human lung cancer cells and a clinical isolate of methicillin-resistant Staphylococcus aureus, respectively. This is the first report of cytotoxic activity for the endiandric/beilschmiedic acid class of compounds.

Isolation of apoptosis-inducing stilbenoids from four members of the Orchidaceae family.

High-throughput natural product research produced a suite of anticancer hits among several species of the Orchidaceae family (Oncidium microchilum, O. isthmi, and Myrmecophila humboldtii). A commercial Oncidium sp. was also examined as a convenient source of additional material. Isolation and structure elucidation led to the identification of fifteen stilbenoids including a new phenanthraquinone and two new dihydrostilbenes. NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7-mm MicroCryoProbe or a 5-µL CapNMR capillary microcoil. Several compounds inhibited proliferation of NCI-H460 and M14 cancer cell lines. All compounds were also examined for their ability to induce apoptosis. Apoptosis induction was determined by measuring caspase 3/7 activation and LDH release in a NCI-H460 cell line. Based on these results, a portion of the extract from a commercially available Oncidium sp. was chemically modified in an attempt to obtain additional phenanthraquinones.

Seasonal changes in VO2max among Division 1A collegiate women soccer players.

Aerobic capacity and body composition were measured at 3 time points over a 1-year period in 26 Division 1A women soccer players from Texas A&M University, in order to determine whether there were seasonal changes in these parameters. Subjects were tested in December, immediately following a 4-month competitive season; in April, following 15 weeks of strength and conditioning; and immediately prior to the start of the regular season in August, following a 12-week summer strength and conditioning program. A periodized strength and conditioning program design was incorporated in order to optimize anaerobic and oxidative capacity immediately prior to the regular competitive season. Significant differences in VO2max were measured between August (49.24 +/- 4.38 ml x kg(-1) x min(-1)) and December (44.87 +/- 4.61 ml x kg(-1) x min(-1)). No significant changes in aerobic capacity were found between April (47.43 +/- 4.01 ml x kg(-1) x min(-1)) and August (49.64 +/- 5.25 ml x kg(-1) x min(-1)). Significant increases in body fat were measured between August (15.71 +/- 2.92%) and December (18.78 +/- 2.79%), before and after the competitive season, respectively. No significant changes in body fat were found between April (16.24 +/- 2.95%) and August (15.71 +/- 2.92%). The results of this study suggest that decreases in muscle mass over the course of a regular competitive season contribute to decreases in aerobic capacity in collegiate women soccer players. Although it is unknown whether this decrease in muscle mass is the result of inadequate training or a normal adaptation to the physiological demands imposed by soccer, the results of the current study suggest that resistance training volume should be maintained during the competitive season, in order to maintain preseason levels of muscle mass.

Escherichia coli from urine of female patients with urinary tract infections is competent for intracellular bacterial community formation.

Nearly 50% of women experience at least one urinary tract infection (UTI) in their lifetime. Studies with mice have revealed that uropathogenic Escherichia coli (UPEC) isolates invade superficial umbrella cells that line the bladder, allowing them to find a safe haven and subvert clearance by innate host responses. Rapid intracellular replication results in the formation of distinctive intracellular bacterial communities (IBCs). In this study, we evaluated whether UPEC strains cultured from the urine of women and classified as causing acute cystitis, recurrent cystitis, asymptomatic bacteriuria, or pyelonephritis could progress through the IBC cascade in a well-characterized mouse model of cystitis. Of 18 UPEC isolates collected from women, 15 formed IBCs. Variations in the size, number, and kinetics of IBC formation were observed with strains isolated from women with different clinical syndromes. Two of the three isolates that did not form IBCs when inoculated alone were able to do so when coinoculated with an isolate that was capable of generating IBCs. The mixed infections dramatically altered the behavior of the coinfecting bacteria relative to their behavior in a single infection. The study also showed that mice with five different genetic backgrounds can support IBC formation. Although UPEC isolates differ genetically in their virulence factors, the majority of UPEC isolates from different types of UTI proceed through the IBC pathway, confirming the generality of IBCs in UTI pathogenesis in mice.

The Helicobacter felis model of adoptive transfer gastritis.

The bacterium Helicobacter pylori is a major human pathogen and the principal cause of acute and chronic gastritis, gastric and duodenal ulcer disease, and gastric adenocarcinoma. Infection with gastric Helicobacter results in an early infiltration of neutrophils, monocytes, and natural killer cells, followed by an influx of T cells and plasma cells. Although the critical components of this gastric infiltrate that lead to disease are unclear, the Helicobacter felis-infected mouse and other mouse models of Helicobacter-associated gastritis have demonstrated the critical nature of adaptive immunity in the development of gastric epithelial pathology. To further investigate the role of adaptive immunity in this disease, adoptive transfer models of disease have also been utilized. These models clearly demonstrate that it is the host CD4+ T lymphocyte response that is crucial for the development of Helicobacter-associated gastric epithelial changes.

Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J mice.

Enterococcus faecalis is a gram-positive bacterium that can cause a variety of nosocomial infections of which urinary tract infections are the most common. These infections can be exceptionally difficult to treat because of drug resistance of many E. faecalis isolates. Despite their troublesome nature, little is known about the host or bacterial factors necessary for E. faecalis to cause disease in the urinary tract. Using a mouse model of urinary tract infection, we have shown that E. faecalis is capable of persisting in the kidneys of mice for at least 2 weeks. In contrast, bacterial titers from the bladders of the same mice were inconsistent and tended to be much lower than those recovered from the kidney. This preference for the kidney over the bladder is also observed in other clinical E. faecalis strains. Histologic examination of bladder and kidney tissues demonstrated that E. faecalis induced an inflammatory response in the kidney but not in the bladder. This inflammatory response was TLR2 independent and did not induce inflammatory markers typically associated with uropathogenic Escherichia coli. Using a competition assay, we demonstrated that a pyelonephritis clinical isolate had a growth advantage over a laboratory strain of E. faecalis in the kidneys but not in the bladders of mice. Taken together, these results demonstrate that E. faecalis has tropism for the kidneys in the urinary tracts of mice and that this system can be used to study factors involved in the pathogenesis of urinary tract infections.

Host subversion by formation of intracellular bacterial communities in the urinary tract.

Urinary tract infections pose a serious health threat with respect to antibiotic resistance and high recurrence rates. While the host robustly responds to bacterial infiltration into the bladder, uropathogenic Escherichia coli can survive the onslaught to persist for months after initially infecting. To accomplish this feat, uropathogenic E. coli forms intracellular bacterial communities, with many biofilm-like properties, within the bladder epithelium. These communities may allow bacteria to subvert host defenses and form a persistent reservoir in the bladder.

Toll-like receptor 4 on stromal and hematopoietic cells mediates innate resistance to uropathogenic Escherichia coli.

Innate host defenses at mucosal surfaces are critical in the early stages of many bacterial infections. In addition to cells of the traditional innate immune system, epithelial cells can also produce inflammatory mediators during an infection. However, the role of the epithelium in innate host defense in vivo is unclear. Recent studies have shown that lipopolysaccharide (LPS) recognition is critical for bladder epithelial cells to recognize and respond to Escherichia coli. Moreover, the LPS-nonresponsive mouse strain C3HHeJ, which has a mutation in the primary LPS receptor, Toll-like receptor 4 (TLR4), is extremely susceptible to infection with uropathogenic strains of E. coli. In this study, a bone marrow transplant approach was used to investigate the specific contributions of the bladder epithelium (and other stromal cells) in the TLR4-mediated innate immune response to the invading E. coli pathogen. Mice expressing the mutant TLR4 in the epithelialstromal compartment were not able to mount a protective inflammatory response to control the early infection even when their hematopoietic cells expressed wild-type TLR4. However, the presence of TLR4(+) epithelialstromal cells was not sufficient to activate an acute inflammatory response unless the hematopoietic cells were also TLR4(+). These results demonstrated that bladder epithelial cells play a critical role in TLR4-mediated innate immunity in vivo during a mucosal bacterial infection.

CD14- and Toll-like receptor-dependent activation of bladder epithelial cells by lipopolysaccharide and type 1 piliated Escherichia coli.

The gram-negative bacterium Escherichia coli is the leading cause of urinary tract infection. The interaction between type 1 piliated E. coli and bladder epithelial cells leads to the rapid production of inflammatory mediators, such as interleukin-6 (IL-6) and IL-8. Conflicting reports have been published in the literature regarding the mechanism by which uroepithelial cells are activated by type 1 piliated E. coli. In particular, the role of lipopolysaccharide (LPS) in these responses has been an area of significant debate. Much of the data arguing against LPS-mediated activation of bladder epithelial cells have come from studies using a renal epithelial cell line as an in vitro model of the urinary epithelium. In this report, we analyzed three bladder epithelial cell lines and demonstrated that they all respond to LPS. Furthermore, the LPS responsivity of the cell lines directly correlated with their ability to generate IL-6 after E. coli stimulation. The LPS receptor complex utilized by the bladder epithelial cell lines included CD14 and Toll-like receptors, and signaling involved the activation of NF-kappaB and p38 mitogen-activated protein kinase. Also, reverse transcription-PCR analysis demonstrated that bladder epithelial cells express CD14 mRNA. Thus, the molecular machinery utilized by bladder epithelial cells for the recognition of E. coli is very similar to that described for traditional innate immune cells, such as macrophages. In contrast, the A498 renal epithelial cell line did not express CD14, was hyporesponsive to LPS stimulation, and demonstrated poor IL-6 responses to E. coli.