Mixed adjuvant formulations reveal a new combination that elicit antibody response comparable to Freund's adjuvants

Adjuvant formulations capable of inducing high titer and high affinity antibody responses would provide a major advance in the development of vaccines to viral infections such as HIV-1. Although oil-in-water emulsions, such as Freund's adjuvant (FCA/FIA), are known to be potent, their toxicity and reactogenicity make them unacceptable for human use. Here, we explored different adjuvants and compared their ability to elicit antibody responses to FCA/FIA. Recombinant soluble trimeric HIV-1 gp140 antigen was formulated in different adjuvants, including FCA/FIA, Carbopol-971P, Carbopol-974P and the licensed adjuvant MF59, or combinations of MF59 and Carbopol. The antigen-adjuvant formulation was administered in a prime-boost regimen into rabbits, and elicitation of antigen binding and neutralizing antibodies (nAbs) was evaluated. When used individually, only FCA/FIA elicited significantly higher titer of nAbs than the control group (gp140 in PBS (p<0.05)). Sequential prime-boost immunizations with different adjuvants did not offer improvements over the use of FCA/FIA or MF59. Remarkably however, the concurrent use of the combination of Carbopol-971P and MF59 induced potent adjuvant activity with significantly higher titer nAbs than FCA/FIA (p<0.05). This combination was not associated with any obvious local or systemic adverse effects. Antibody competition indicated that the majority of the neutralizing activities were directed to the CD4 binding site (CD4bs). Increased antibody titers to the gp41 membrane proximal external region (MPER) and gp120 V3 were detected when the more potent adjuvants were used. These data reveal that the combination of Carbopol-971P and MF59 is unusually potent for eliciting nAbs to a variety of HIV-1 nAb epitopes.     

Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis

Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.     

Metabolism of 17α-methyl-5α-dihydrotestosterone in the rabbit

17α-Methyl-5α-dihydrotestosterone and the reduced metabolites, 17α-methyl-5α-androstane-3α, 17β-diol and -3β, 17β-diol together with two hydroxylated metabolites, 17α-methyl-5α-androstane-3β, 15α, 17β-triol and 17α-methyl-5α-androstane-3α, 6α, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5α-dihydrotestosterone. Formation of the C-6 hydroxylated derivative demonstrates that the 4,6-enolization of a 4-en-3-one is not a necessary requirement for hydroxylation at C-6 of the androstane nucleus in the rabbit. No evidence was obtained for the presence of 17α-methyl/17β-hydroxyl epimerization.     

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.     

A eudesmane acid from Montanoa speciosa

The major constituent of a leaf-surface methylene dichloride wash of Montanoa speciosa was a eudesmane acid, 1,2-dehydro-3-oxo-costic acid.     

Light modulation of postural flexor motoneurones in the tail-fan of the squat lobsterGalathea strigosa (Crustacea,Anomura)

Summary Photosensitivity in the terminal abdominal ganglion (G5) of an anomuran, the squat lobsterGalathea strigosa (Crustacea, Decapoda, Anomura), is described. In contrast to the caudal photoreceptors (CPRs) of long-tailed natantid and macruran decapod crustaceans, the caudal photosensitive elements in G5 inG. strigosa apparently lack the conventional spiking rostral conduction pathways to the thoracic ganglia, and instead make their output connections to a bilateral pair of tonic flexor motoneurones originating within the caudal ganglion itself. These flexor motoneurones modulate the activity of two bilaterally paired uropod coxopodite tonic flexor muscles. This photomodulated motoneurone (PMMN) activity is not abolished by sectioning the abdominal nerve cord anterior to G5. The pattern of photosensitivity, while differing from that shown by other CPRs, resembles instead the pattern attributed to photosensitive interneurones (PSIs) of rostral abdominal ganglia of crayfish and other long-tailed decapod crustaceans.The caudal PSIs inG. strigosa appear to be involved in the postural control of the tail-fan as it is held flexed against the cephalothorax.     

Six new melampolides from Tetragonotheca helianthoides

Chemical analysis of Tetragonotheca helianthoides L. yielded six new melampolide-type sesquiterpene lactones, tetrahelins A–F. All new compounds have the same medium ring skeleton and differ by the ester moieties at C-8 and C-9. Tetrahelin A and B contain the side chain 2-methyl-2,3-diacetoxybutanoate and tetrahelin E 3-hydroxy-3-methylbutanoate. Both ester side chains have previously not been found in sesquiterpene lactones.     

A rearing system for colostrum-deprived neonatal piglets

A system for the artificial rearing of colostrum-deprived neonatal piglets is described. A total of 443 piglets in 6 batches was reared. Each piglet, immediately after birth was placed in a separate incubator which was ventilated with heated filtered air. Piglets were fed hourly by an automatic system and were offered a liquid milk substitute. The mean survival rate up to the time piglets were transferred out of the rearing unit at 2 weeks of age was 78 +/- 7%. The mean daily live weight gain up to 2 weeks of age was 148 +/- 16 g/day and the dry matter intake over the same period was 154 +/- 10 g/day per piglet.