Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.     

Nudibranch predation and dietary preference for the polyps of <Emphasis Type="Italic">Aurelia</Emphasis><Emphasis Type="Italic">labiata</Emphasis> (Cnidaria: Scyphozoa)

There is concern that jellyfish blooms may be increasing worldwide. Some factors controlling population size, such as temperature and food, often have been studied; however, the importance of predators is poorly known. Aeolid nudibranchs feed on cnidarians, but their predation on the benthic polyps of scyphozoan rarely has been documented. To understand the potential of nudibranchs to consume polyps, we tested several predation preference hypotheses with the generalist feeding nudibranch, Hermissenda crassicornis, and polyps of the common moon jellyfish, Aurelia labiata. Of the six prey species tested during feeding experiments, A. labiata polyps and the tunicate Distaplia occidentalis were significantly preferred. Nudibranch size, diurnal cycle, and ingestive conditioning did not significantly influence prey choice. Nudibranchs showed significant positive chemotaxis toward living polyps, hydroids, and tunicates, but not to sea anemones. Nudibranch chemotaxis was significantly more positive to polar extract of A. labiata than of D. occidentalis. Consumption of polyps was correlated with nudibranch size, with mean consumption by large nudibranchs (>0.92 g) of about 31 polyps h⁻¹. Three other nudibranch species also ate A. labiata polyps. Our results emphasize the potential importance of predation for controlling jellyfish benthic polyp populations and consequent jellyfish blooms.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Campylobacter jejuni is a Gram‐negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain‐Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram‐negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O‐antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐glucose and dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′‐phosphate dependent aminotransferase. Here, we describe the first three‐dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP‐3‐amino‐3,6‐dideoxy‐d ‐galactose or dTDP‐3‐amino‐3,6‐dideoxy‐d ‐glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C‐4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.     

Genetic Determinants of Circadian Rhythmicity in Neurospora

Timex, a strain of Neurospora crassa which exhibits a circadian rhythm of conidia formation in growth-tube cultures, has been found to differ from wild-type strains by two genes. One gene, inv, is responsible for an invertase deficiency, whereas the second gene, bd, is of unknown function. Both genes map independently from other genes known to induce Neurospora rhythmicity. The inv gene is not essential for the timex phenotype because bd strains express that phenotype on certain media. Although inv strains do exhibit some rhythmicity of their own, the rhythmicity apparently is not a direct result of the invertase deficiency, since there is no correlation between invertase level and rhymicity in 29 strains tested. Of the 29 strains tested, 20 exhibited some rhythmicity in growth-tube cultures, suggesting that morphological manifestations of rhythmicity in Neurospora may result from the function or the loss of function of numerous genes, or both. There was no correlation in these strains between rhythmicity and (i) genetic background; (ii) geographical origin; or (iii) nutritional requirements.     

Design and synthesis of potent, orally-active DGAT-1 inhibitors containing a dioxino[2,3-d]pyrimidine core

A novel series of potent DGAT-1 inhibitors was developed originating from the lactam-based clinical candidate PF-04620110. Incorporation of a dioxino[2,3-d]pyrimidine-based core afforded good alignment of pharmacophore features and resulted in improved passive permeability. Development of an efficient, homochiral synthesis of these targets facilitated confirmation of predictions regarding the stereochemical-dependence of DGAT-1 inhibition for this series. Compound 10 was shown to be a potent inhibitor of human DGAT-1 (10 nM) and to suppress triglyceride synthesis at oral doses of <3mg/kg.     

Circadian Nature of a Rhythm Expressed by an Invertaseless Strain of Neurospora crassa

A new strain of Neurospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth tube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is about 24 hours (22.7 hours at 25 degrees ); B) the period is relatively temperature-independent (Q(10) is between 0.95 and 1.21 for temperature range of 18 to 35 degrees ); C) the rhythm persists in continuous darkness at constant temperature for a minimum of 14 days without damping out; and D) the phase of the rhythm can be shifted by a single brief exposure to light. The sensitivity of this strain to light has been demonstrated further by the entrainment of the rhythm to a period of 24.0 hours using a suitable light-dark regime, and by the inhibition by light of the appearance of a rhythm; i.e., continuous conidiation occurs when the strain is subjected to continuous light. The new strain is compared to 2 other strains of Neurospora which also express a rhythm, patch and clock.