The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2^b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2^b) (B6) and allogeneic C3H/Hej (H-2^k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.     

Adhesion of Vancomycin-Resistant Enterococcus to Human Intestinal Mucus

The intestinal mucus layer provides a potential niche for colonization by vancomycin-resistant Enterococcus faecium (VREF). We therefore examined the ability of six VREF strains to adhere to human intestinal mucus and determined binding kinetics. Four of six (67%) VREF strains demonstrated significant adhesion to immobilized intestinal mucus compared with a Salmonella typhimurium–negative control strain, but the level of adherence was low compared with Lactobacillus rhamnosus GG. Binding kinetics studies demonstrated that the maximum number of these four VREF strains that could adhere to a unit surface area of immobilized mucus was similar to or higher than the maximum number of L. rhamnosus GG that could adhere; however, L. rhamnosus GG demonstrated 20- to 130-times higher affinity than the VREF strains. These results demonstrate that VREF strains may adhere to human intestinal mucus and suggest that L. rhamnosus GG might be able to displace VREF strains.     

Taxonomie und Systematik der HippocardioideaPojeta & Runnegar, 1976 [n. superfam.] (Mollusca; Rostroconchia)

The revisions of the families PseudobigaleaidaeHoare, Mapes &; Yancey, 2002 and HippocardiidaePojeta &; Runnegar, 1976 led to the introduction of a new taxon at the superfamily rank in order to unite the two families and distinguish them from the members of the Conocardiidae and Bransoniidae within the order Conocardiida. The autapomorphy of this new superfamily HippocardioideaPojeta &; Runnegar, 1976 [n. superfam.] — the more or less uniform development of at least one hood — is considered taxonomically as so important that we exclude a common ancestry of the almost contemporaneously occurring taxa of the Hippocardiidae, Pseudobigaleaidae on the one hand and the Bransoniidae sensuPojeta &; Runnegar on the other. The family Hippocardiidae is divided into six taxa on subfamily rank, the HippocardiinaePojeta &; Runnegar, Babinicardiinae n. subfam., Barrandeicardiinae n. subfam., Bohemicardiinae n. subfam., Goticardiinae n. subfam. and Hassiacardiinae n. subfam. At the species level, 13 taxa are assigned to the generaBabinicardia n. gen.,Barrandeicardia n. gen.,Bohemicardia n. gen.,Filicardia n. gen,.Globocardia n. gen.,Goticardia n. gen.,Pohlia n. gen. andHassiacardia n. gen. Additionally, the speciesGoticardia kauffmannii n. gen. n. sp. is introduced, whereas the primary homonymy of ?Conocardium“reticulatum Babin, 1966 is retained, because this taxon remained inassignable to any of the existing genera, but is considered as a member of the Hippocardiidae. The revised family Hippocardiidae occurred stratigraphically from the Middle Silurian (Wenlockian) until Mississippian time; members of the new superfamily Hippocardioidea are known from the Ordovician until the Pennsylvanian.     

Mandibular size and shape variation in the hominins at Dmanisi, Republic of Georgia

The hominin fossils of Dmanisi, Republic of Georgia, present an ideal means of assessing levels of skeletal size and shape variation in a fossil hypodigm belonging to the genus Homo because they have been recovered from a spatially and temporally restricted context. We compare variation in mandible size and shape at Dmanisi to that of extant hominoids and extinct hominins. We use height and breadth measurements of the mandibular corpus at the first molar and the symphysis to assess size, and analyze shape based on size-adjusted (using a geometric mean) versions of these four variables. We compare size and shape variation at Dmanisi relative to all possible pairs of individuals within each comparative taxon using an exact resampling procedure of the ratio of D2600 to D211 and the average Euclidean distance (AED) between D2600 and D211, respectively. Comparisons to extant hominoids were conducted at both the specific and subspecific taxonomic levels and to extinct hominins by adopting both a more, and less speciose, hominin taxonomy. Results indicate that the pattern of variation for the Dmanisi hominins does not resemble that of any living species: they exhibit significantly more size variation when compared to modern humans, and they have significantly more corpus shape variation and size variation in corpus heights and overall mandible size than any extant ape species. When compared to fossil hominins they are also more dimorphic in size (although this result is influenced by the taxonomic hypothesis applied to the hominin fossil record). These results highlight the need to re-examine expectations of levels of sexual dimorphism in members of the genus Homo and to account for marked size and shape variation between D2600 and D211 under the prevailing view of a single hominin species at Dmanisi.     

Natural and experimental West Nile virus infection in five raptor species

We studied the effects of natural and/or experimental infections of West Nile virus (WNV) in five raptor species from July 2002 to March 2004, including American kestrels (Falco sparverius), golden eagles (Aquila chrysaetos), red-tailed hawks (Buteo jamaicensis), barn owls (Tyto alba), and great horned owls (Bubo virginianus). Birds were infected per mosquito bite, per os, or percutaneously by needle. Many experimentally infected birds developed mosquito-infectious levels of viremia (>10(5) WNV plaque forming units per ml serum) within 5 days postinoculation (DPI), and/ or shed virus per os or per cloaca. Infection of organs 15-27 days postinoculation was infrequently detected by virus isolation from spleen, kidney, skin, heart, brain, and eye in convalescent birds. Histopathologic findings varied among species and by method of infection. The most common histopathologic lesions were subacute myocarditis and encephalitis. Several birds had a more acute, severe disease condition represented by arteritis and associated with tissue degeneration and necrosis. This study demonstrates that raptor species vary in their response to WNV infection and that several modes of exposure (e.g., oral) may result in infection. Wildlife managers should recognize that, although many WNV infections are sublethal to raptors, subacute lesions could potentially reduce viability of populations. We recommend that raptor handlers consider raptors as a potential source of WNV contamination due to oral and cloacal shedding.     

Hematology, serum chemistry, and serology of Galápagos penguins (Spheniscus mendiculus) in the Galápagos Islands, Ecuador

The Galápagos penguin (Spheniscus mendiculus) is an endangered species endemic to the Galápagos Islands, Ecuador. In 2003 and 2004, 195 penguins from 13 colonies on the islands of Isabela and Fernandina in the Galápagos archipelago were examined. Genetic sexing of 157 penguins revealed 62 females and 95 males. Hematology consisted of packed cell volume (n = 134), white blood cell differentials (n = 83), and hemoparasite blood smear evaluation (n = 114). Microfilariae were detected in 22% (25/114) of the blood smears. Female penguins had significantly higher eosinophil counts than males. Serum chemistry on 83 penguins revealed no significant differences between males and females. Birds were seronegative to avian paramyxovirus type 1-3, avian influenza virus, infectious bursal disease virus, Marek's disease virus (herpes), reovirus, avian encephalomyelitis virus, and avian adenovirus type 1 and 2 (n = 75), as well as to West Nile virus (n = 87), and Venezuelan, western and eastern equine encephalitis viruses (n = 26). Seventy-five of 84 (89%) penguins had antibodies to Chlamydophila psittaci but chlamydial DNA was not detected via polymerase chain reaction in samples from 30 birds.     

Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.     

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1

Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain G?1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes.     

IgE-dependent activation of sphingosine kinases 1 and 2 and secretion of sphingosine 1-phosphate requires Fyn kinase and contributes to mast cell responses

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells results in the production and secretion of sphingosine 1-phosphate (S1P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of sphingosine kinase (SphK1 and SphK2) are expressed and activated upon FcepsilonRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcepsilonRI coupling to SphK1 and -2 and for subsequent S1P production. Normal activation of SphK1 and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK1 and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK1 activation differed from that of SphK2. Whereas SphK1 was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to FcepsilonRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.