Repeated concanavalin A challenge in mice induces an interleukin 10-producing phenotype and liver fibrosis.

Weekly injections of Concanavalin A (Con A) were performed in BALB/c mice to evaluate the pattern of cytokine production and liver injury. High serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-gamma) were found in the serum after the first 2 injections of Con A but rapidly decreased from the third injection. Conversely, IL-10 serum levels after repeated Con A challenge increased by 7 times from week 1 to 20. In vivo depletion studies indicated that CD4(+) T cells are essential in IL-10 production. Hepatocyte necrosis was only observed after the first injections of Con A whereas centrilobular inflammatory infiltrates persisted up to 20 weeks. Perisinusoidal liver fibrosis was also increasingly detected in BALB/c mice, whereas no fibrous change was observed in nude mice after 6 weeks of Con A challenge. The number of stellate cells, detected by immunostaining, increased after 20 weeks of Con A injections. Liver cytokine messenger RNA (mRNA) expression after 20 weeks showed expression of transforming growth factor beta1 (TGF-beta1), IL-10, and IL-4 whereas IL-2 was no more expressed. The present study shows that mice repeatedly injected with Con A develop liver fibrosis. The cytokine-release pattern observed after 1 injection of Con A is rapidly shifted towards an immunomodulatory phenotype characterized by the systemic production of large amounts of IL-10.

Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis.

BACKGROUND AND AIMS: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to act as a negative regulator of T cell function and has been implicated in the regulation of T helper 1 (Th1)/Th2 development and the function of regulatory T cells. Tests were carried out to determine whether anti-CTLA-4 treatment would alter the polarisation of naive T cells in vivo. METHODS: Mice were treated with anti-CTLA-4 monoclonal antibody (mAb) (UC10-4F10) at the time of immunisation or colonic instillation of trinitrobenzene sulfonic acid (TNBS). The cytokines produced by lymph node cells after in vitro antigenic stimulation and the role of indoleamine 2,3 dioxygenase (IDO) and of interleukin-10 (IL-10) were tested, and the survival of mice was monitored. RESULTS: Injection of anti-CTLA-4 mAb in mice during priming induced the development of adaptive CD4(+) regulatory T cells which expressed high levels of ICOS (inducible co-stimulator), secreted IL-4 and IL-10. This treatment inhibited Th1 memory responses in vivo and repressed experimental intestinal inflammation. The anti-CTLA-4-induced amelioration of disease correlated with IDO expression and infiltration of ICOS(high) Foxp3(+) T cells in the intestine, suggesting that anti-CTLA-4 acted indirectly through the development of regulatory T cells producing IL-10 and inducing IDO. CONCLUSIONS: These observations emphasise the synergy between IL-10 and IDO as anti-inflammatory agents and highlight anti-CTLA-4 treatment as a potential novel immunotherapeutic approach for inducing adaptive regulatory T cells.

Magnitude and breadth of a nonprotective neutralizing antibody response in an efficacy trial of a candidate HIV-1 gp120 vaccine.

BACKGROUND: A candidate vaccine consisting of human immunodeficiency virus type 1 (HIV-1) subunit gp120 protein was found previously to be nonprotective in an efficacy trial (Vax004) despite strong antibody responses against the vaccine antigens. Here we assessed the magnitude and breadth of neutralizing antibody responses in Vax004. METHODS: Neutralizing antibodies were measured against highly sensitive (tier 1) and moderately sensitive (tier 2) strains of HIV-1 subtype B in 2 independent assays. Vaccine recipients were stratified by sex, race, and high versus low behavioral risk of HIV-1 acquisition. RESULTS: Most vaccine recipients mounted potent neutralizing antibody responses against HIV-1(MN) and other tier 1 viruses. Occasional weak neutralizing activity was detected against tier 2 viruses. The response against tier 1 and tier 2 viruses was significantly stronger in women than in men. Race and behavioral risk of HIV-1 acquisition had no significant effect on the response. Prior vaccination had little effect on the neutralizing antibody response that arose after infection. CONCLUSIONS: Weak overall neutralizing antibody responses against tier 2 viruses is consistent with a lack of protection in this trial. The magnitude and breadth of neutralization reported here should be useful for identifying improved vaccines.

Fiber-optic interferometric two-dimensional scattering-measurement system.

We present a fiber-optic interferometric system for measuring depth-resolved scattering in two angular dimensions using Fourier-domain low-coherence interferometry. The system is a unique hybrid of the Michelson and Sagnac interferometer topologies. The collection arm of the interferometer is scanned in two dimensions to detect angular scattering from the sample, which can then be analyzed to determine the structure of the scatterers. A key feature of the system is the full control of polarization of both the illumination and the collection fields, allowing for polarization-sensitive detection, which is essential for two-dimensional angular measurements. System performance is demonstrated using a double-layer microsphere phantom. Experimental data from samples with different sizes and acquired with different polarizations show excellent agreement with Mie theory, producing structural measurements with subwavelength accuracy.

A kinesin motor in a force-producing conformation.

BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.

Co-orientation of replication and transcription preserves genome integrity.

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over approximately 1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

R5 clade C SHIV strains with tier 1 or 2 neutralization sensitivity: tools to dissect env evolution and to develop AIDS vaccines in primate models.

BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.

Cryptococcal cell morphology affects host cell interactions and pathogenicity.

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.

Interaction between 3’,5’-cyclic monophosphate, volume-regulated anion channels and the Na+-HCO3- cotransporter NBCe1 in the regulation of nutrient- and hypotonicity-induced insulin release from rat pancreatic islets and tumoral insulin-producing BRIN-BD11 cells

info:eu-repo/semantics/published

Effects of the inhibitor of anoctamin 1, tannic acid, on insulin-producing cells

info:eu-repo/semantics/submittedForPublication

Antisense Gene Silencing and Bacteriophages as Novel Disinfection Processes for Engineered Systems

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

Interleukin-15 receptor blockade in non-human primate kidney transplantation.

BACKGROUND: Interleukin (IL)-15 is a chemotactic factor to T cells. It induces proliferation and promotes survival of activated T cells. IL-15 receptor blockade in mouse cardiac and islet allotransplant models has led to long-term engraftment and a regulatory T-cell environment. This study investigated the efficacy of IL-15 receptor blockade using Mut-IL-15/Fc in an outbred non-human primate model of renal allotransplantation. METHODS: Male cynomolgus macaque donor-recipient pairs were selected based on ABO typing, major histocompatibility complex class I typing, and carboxy-fluorescein diacetate succinimidyl ester-based mixed lymphocyte responses. Once animals were assigned to one of six treatment groups, they underwent renal transplantation and bilateral native nephrectomy. Serum creatinine level was monitored twice weekly and as indicated, and protocol biopsies were performed. Rejection was defined as a increase in serum creatinine to 1.5 mg/dL or higher and was confirmed histologically. Complete blood counts and flow cytometric analyses were performed periodically posttransplant; pharmacokinetic parameters of Mut-IL-15/Fc were assessed. RESULTS: Compared with control animals, Mut-IL-15/Fc-treated animals did not demonstrate increased graft survival despite adequate serum levels of Mut-IL-15/Fc. Flow cytometric analysis of white blood cell subgroups demonstrated a decrease in CD8 T-cell and natural killer cell numbers, although this did not reach statistical significance. Interestingly, two animals receiving Mut-IL-15/Fc developed infectious complications, but no infection was seen in control animals. Renal pathology varied widely. CONCLUSIONS: Peritransplant IL-15 receptor blockade does not prolong allograft survival in non-human primate renal transplantation; however, it reduces the number of CD8 T cells and natural killer cells in the peripheral blood.

Rhyme and Reason: Analyses of Dual Retrieval Cues

If and only if each single cue uniquely defines its target, a independence model based on fragment theory can predict the strength of a combined dual cue from the strengths of its single cue components. If the single cues do not each uniquely define their target, no single monotonic function can predict the strength of the dual cue from its components; rather, what matters is the number of possible targets. The probability of generating a target word was .19 for rhyme cues, .14 for category cues, and .97 for rhyme-plus-category dual cues. Moreover, some pairs of cues had probabilities of producing their targets of .03 when used individually and 1.00 when used together, whereas other pairs had moderate probabilities individually and together. The results, which are interpreted in terms of multiple constraints limiting the number of responses, show why rhymes, which play a minimal role in laboratory studies of memory, are common in real-world mnemonics.