CAF01 potentiates immune responses and efficacy of an inactivated influenza vaccine in ferrets

Trivalent inactivated vaccines (TIV) against influenza are given to 350 million people every year. Most of these are non-adjuvanted vaccines whose immunogenicity and protective efficacy are considered suboptimal. Commercially available non-adjuvanted TIV are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6'-dibehenate, CAF01) was developed. CAF01 has proven to enhance both humoral and cell-mediated immune responses to a number of different experimental vaccine candidates. In this study, we compared the immune responses in ferrets to a commercially available TIV with the responses to the same vaccine mixed with the CAF01 adjuvant. Two recently circulating H1N1 viruses were used as challenge to test the vaccine efficacy. CAF01 improved the immunogenicity of the vaccine, with increased influenza-specific IgA and IgG levels. Additionally, CAF01 promoted cellular-mediated immunity as indicated by interferon-gamma expressing lymphocytes, measured by flow cytometry. CAF01 also enhanced the protection conferred by the vaccine by reducing the viral load measured in nasal washes by RT-PCR. Finally, CAF01 allowed for dose-reduction and led to higher levels of protection compared to TIV adjuvanted with a squalene emulsion. The data obtained in this human-relevant challenge model supports the potential of CAF01 in future influenza vaccines.     

Organstruktur und Zellfunktion in explantiertem und später auto-retransplantiertem Schilddrüsengewebe

Zusammenfassung Schilddrüsengewebe von erwachsenen Kaninchen wurde in heterologem Medium in Rollröhrchen gezüchtet und 3 Wochen bis 4 1/2 Monate später auf das Spenderkaninchen rückverpflanzt. Hier blieben die Transplantate von 1 bis zu 8 Monaten. Die histologischen Befunde unmittelbar vor und nach Abschluß der Transplantation wurden miteinander verglichen. Unter beiden Lebensbedingungen, in vitro und im Transplantat, produzierten die Schilddrüsenzellen massenhaft Sekret, das in seinem färberischen Verhalten dem Schilddrüsenkolloid gleicht. Es wird für unwahrscheinlich gehalten, daß das Sekret biochemisch vollwertiges Schilddrüsenkolloid darstellt. Das Sekret wurde in großen Mengen intrazellulär gestapelt und führte schließlich zum Zellverfall.In den Transplantaten bestand nur dann Follikelanordnung, wenn diese auch noch in der Ausgangskultur vorhanden war. Die Follikel waren in jungen Transplantaten zunächst recht gut von Kapillaren umsponnen, doch verödeten diese später wieder. Alte Transplantate gingen schließlich genau so wie die gefäßlosen Gewebekulturen und wie alte, von vorn herein gefäßlos bleibende Transplantate durch intrazelluläre Sekretstapelung zugrunde. Es wird vermutet, daß in vitro der Mangel an thyreotropem Hormon zu dieser Fehlsteuerung führte und daß die Zellen auch im Transplantat nicht mehr auf das nun zur Verfügung stehende thyreotrope Hormon mit Ausschleusung des Sekrets reagieren konnten, weil dieser Mechanismus schon vorher in vitro pathologisch verändert worden war.Durch die Vorzüchtung wurde das Autotransplantationsergebnis erheblich verschlechtert.Eine maligne Entartung trat während der Züchtung in vitro nicht ein.     

Appomattoxia ancistrophora gen. et sp. nov., a new Early Cretaceous plant with similarities to Circaeaster and extant Magnoliidae

A new genus and species of fossil angiosperm (Appomattoxia ancistrophora) is established based on well-preserved fruiting units and associated pollen from the Early Cretaceous (Early or Middle Albian) Puddledock locality in the Potomac Group sequence of Virginia, eastern North America. Fruiting units are small, unilocular, and with a single, pendulous, orthotropous seed. The fruit surface is characterized by densely spaced unicellular spines with hooklike tips, which probably functioned in biotic dispersal. Pollen grains adhering to the stigmatic area of many specimens are monocolpate and tectate with granular to columellate infratectal structure, and are similar to dispersed grains assigned to Tucanopollis and Transitoripollis. Comparison of fossil Appomattoxia ancistrophora with extant plants reveals an unusual combination of characters that includes similarities with some magnoliid taxa, particularly Piperales (Piperaceae, Saururaceae) and Laurales (Chloranthaceae), as well as the monotypic ranunculid family Circaeasteraceae. Appomattoxia ancistrophora differs from extant Piperales in having a pendulous rather than erect ovule, and differs from extant Circaeaster in details of the fruit wall, as well as the presence of monosulcate rather than tricolpate pollen.     

High- and low-affinity GABA-receptors in cultured cerebellar granule cells regulate transmitter release by different mechanisms

The ability of high- and low-affinity GABA_A-receptors, respectively to inhibit depolarization coupled transmitter release was studied in cultured glutamatergic cerebellar granule cells which, depending on the culture conditions, express either high-affinity GABA_A-receptors alone or high-affinity receptors together with low-affinity receptors. In order to gain information about the coupling of these receptors to chloride channels the effect of picrotoxin and binding of [³⁵S]t-butylbicyclophosphorothionate, both of which interact specifically with such channels were studied. Moreover, the influence of Flunitrazepam on the GABA-mediated inhibition of transmitter release was investigated to see if the GABA-receptors are coupled to benzodiazepine binding sites. Under conditions where the granule cells express only high-affinity GABA_A-receptors it was found that GABA was able to inhibit transmitter release elicited by mild depolarization induced either by 30 mM KCl or 25 μM glutamate. This effect of GABA could be enhanced by Flunitrazepam and blocked by picrotoxin. However, transmitter release from these neurones induced by a more pronounced depolarization (55 mM KCl) could not be inhibited by GABA. Under conditions where the neurons express both high- and low-affinity GABA_A-receptors transmitter release elicited by 55 mM KCl could be inhibited by GABA but this inhibitory effect of GABA could not be blocked by picrotoxin, nor could it be enhanced by Flunitrazepam. These results strongly suggest that while the action of the high-affinity GABA_A-receptors is coupled to chloride channels and benzodiazepine binding sites, the physiological action of the low-affinity GABA_A-receptors is not. This lack of coupling between the low-affinity GABA_A-receptors and chloride channels is further supported by the finding that the K_D and B_max values for [³⁵S]TBPS binding to the granule cells were independent of whether or not the cells expressed low-affinity GABA_A-receptors. While the results clearly show that the inhibitory action of GABA mediated by low-affinity GABA_A-receptors is not coupled to chloride channels, the exact mechanism of action of these receptors still remains to be elucidated.     

Phylogeny and taxonomy of Macrolepiota (Agaricaceae)

The position and composition of Macrolepiota within the Agaricaceae and its phylogenetic relationships with other members of the family were investigated, using both molecular (ITS and LSU rDNA sequences) and morphological characters. The molecular data separate the genus into two clades. The first clade comprises M. procera, M. mastoidea, M. clelandii and allies and is a sister group of Leucoagaricus and Leucocoprinus. The second, more diverse, clade, with M. rachodes and allies, M. globosa, Chlorophyllum molybdites, Leucoagaricus hortensis and Endoptychum agaricoides, is a sister group of Agaricus. The separation of the two clades is supported by morphological characters, such as the structure of the pileus covering, the stipitipellis and the shape of the germ pore and the spore apex. The two clades are regarded as genera for which the names Macrolepiota and Chlorophyllum are proposed. Macrolepiota nympharum does not belong to either clade but is assigned to the genus Leucoagaricus, close to L. leucothites. Endoptychum depressum is transferred to the genus Agaricus as A. inapertus.     

hTERT mRNA dendritic cell vaccination: complete response in a pancreatic cancer patient associated with response against several hTERT epitopes

Immunotherapy targeting the hTERT subunit of telomerase has been shown to induce robust immune responses in cancer patients after vaccination with single hTERT peptides. Vaccination with dendritic cells (DCs) transfected with hTERT mRNA has the potential to induce strong immune responses to multiple hTERT epitopes and is therefore an attractive approach to more potent immunotherapy. Blood samples from such patients provide an opportunity for identification of new, in vivo processed T-cell epitopes that may be clinically relevant. A 62-year-old female patient underwent radical surgery for a pancreatic adenocarcinoma. After relapse, she obtained stable disease on gemcitabine treatment. Due to severe neutropenia, the chemotherapy was terminated. The patient has subsequently been treated with autologous DCs loaded with hTERT mRNA for 3 years. Immunomonitoring was performed at regular intervals following start of vaccination and clinical outcome measured by CT and PET/CT evaluation. The patient developed an immune response against several hTERT-derived Th and CTL epitopes. She presently shows no evidence of active disease based on PET/CT scans. No serious adverse events were experienced and the patient continues to receive regular booster injections. We here provide evidence for the induction of hTERT-specific immune responses following vaccination of a pancreas cancer patient with DCs loaded with hTERT mRNA. These responses are associated with complete remission. A thorough analysis of this patient immune response has provided a unique opportunity to identify novel epitopes, associated with clinical effects. These will be included in future hTERT vaccines.     

Novel mitochondrial creatine transport activity. Implications for intracellular creatine compartments and bioenergetics

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.     

When Earth started blooming: insights from the fossil record

Recent palaeobotanical studies have greatly increased the quantity and quality of information available about the structure and relationships of Cretaceous angiosperms. Discoveries of extremely well preserved Cretaceous flowers have been especially informative and, combined with results from phylogenetic analyses of extant angiosperms (based mainly on molecular sequence data), have greatly clarified important aspects of early angiosperm diversification. Nevertheless, many questions still persist. The phylogenetic origin of the group itself remains as enigmatic as ever and, in some cases, newly introduced techniques from molecular biology have given confusing results. In particular, relationships between the five groups of extant seed plants remain uncertain, and it has sometimes proved difficult to reconcile estimates of the time of divergence between extant lineages made using a 'molecular clock' with the fossil record. One result, however, is becoming increasingly clear: a great deal of angiosperm diversity is extinct. Some groups of angiosperms were evidently more diverse in the past than they are today. In other cases, fossils defy assignment to extant groups at the family level or below. This raises the possibility that evolutionary conclusions based solely upon extant taxa that are merely relics of groups that were once much more diverse might be misled by the effects of extinction. It also introduces the possibility that some early enigmatic fossils might represent lineages that diverged from the main line of angiosperm evolution below the most recent common ancestor of all extant taxa. These, and other questions, are among those that need to be addressed by future palaeobotanical research.     

Studies on curcumin and curcuminoids. XXIX. Photoinduced cytotoxicity of curcumin in selected aqueous preparations.

Natural curcumin was evaluated as a potential photosensitizer for oral applications. The photocytotoxicity of curcumin on salivary gland acinar cells (SM 10-12) was investigated in five aqueous preparations consisting of 5% DMSO, non-ionic micelles, cyclodextrin, liposomes, or a hydrophilic polymer. The difference in phototoxic effects between natural curcumin and synthetic curcumin was examined. Cytotoxicity in SM 10-12 cells exposed to curcumin in the concentration range 0.4-13.5 microM was investigated by MTT test, a fluorescence-staining microscopic test, and by Western immunoblotting techniques. The potential formation of a photoreaction product, hydrogen peroxide, was evaluated by a fluorescence assay. The light source was a halogen lamp used in the dental clinic, emitting mainly in the blue part of the spectrum. The phototoxic effect on SM 10-12 cells was dependent on curcumin concentration, the light dose and the type of preparation. Natural and synthetic curcumin induced phototoxicity to the same extent. Significant effects on the cells were obtained at low curcumin concentrations (< or =0.5 microM) and at a low light dose (< or =6 J cm(-2)), after 3 h incubation. Neither the activation of caspases-3, -7, -8 or -9, nor the formation of hydrogen peroxide could be detected in cells exposed to curcumin and light. The liposome preparation was the most efficient vehicle for curcumin to induce cell death. The phototoxic effect induced by curcumin is highly dependent on the type of preparation. Curcumin might be a potential photosensitizer in the treatment of oral lesions and cancers provided careful selection of the vehicle.