Vaccine-Elicited CD8+ T Cells Protect against Respiratory Syncytial Virus Strain A2-Line19F-Induced Pathogenesis in BALB/c Mice

CD8⁺ T cells may contribute to vaccines for respiratory syncytial virus (RSV). Compared to CD8⁺ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8⁺ T cells are less well defined. We used a peptide vaccine to test the hypothesis that vaccine-elicited RSV-specific CD8⁺ T cells are protective against RSV pathogenesis. BALB/c mice were treated with a mixture (previously termed TriVax) of an M2_82-90 peptide representing an immunodominant CD8 epitope, the Toll-like receptor (TLR) agonist poly(I·C), and a costimulatory anti-CD40 antibody. TriVax vaccination induced potent effector anti-RSV CD8⁺ cytotoxic T lymphocytes (CTL). Mice were challenged with RSV strain A2-line19F, a model of RSV pathogenesis leading to airway mucin expression. Mice were protected against RSV infection and against RSV-induced airway mucin expression and cellular lung inflammation when challenged 6 days after vaccination. Compared to A2-line19F infection alone, TriVax vaccination followed by challenge resulted in effector CD8⁺ T cells with greater cytokine expression and the more rapid appearance of RSV-specific CD8⁺ T cells in the lung. When challenged 42 days after TriVax vaccination, memory CD8⁺ T cells were elicited with RSV-specific tetramer responses equivalent to TriVax-induced effector CD8⁺ T cells. These memory CD8⁺ T cells had lower cytokine expression than effector CD8⁺ T cells, and protection against A2-line19F was partial during the memory phase. We found that vaccine-elicited effector anti-RSV CD8⁺ T cells protected mice against RSV infection and pathogenesis, and waning protection correlated with reduced CD8⁺ T cell cytokine expression.     

Relationship of the sarcoplasmic reticulum to fibril and triadic junction development in skeletal muscle fibers of fetal monkeys and humans.

Examinations of stages of fibril development in muscle fibers of seven Rhesus monkey and six human fetuses reveal SR tubules encircling the Z lines at all stages of fibril development. The encircling SR tubules are continuous with the SR network of tubules which is found surrounding fibrils at all stages of development observed. The SR tubules encircling the Z lines show connections (electron-opaque strands) with the Z lines. The developing triadic junction shows a progressive increase in complexity of structures within the junction. First, membranes of T and SR become apposed with no visible structure between them- Second, tenuous connections are found traversing the space between apposed membranes. Third, well developed bridges are seen traversing the space. And finally, an intermediate density midway between the apposed membranes and parallel to them is found in favorable sections. Junctions between T tubule membranes were also observed and the structures in these junctions are somewhat similar to those found in junctions between T and SR membranes. The change in orientation of triads from predominantly longitudinal to predominantly transverse is complete in the 18-week monkey fetus and incomplete in the latest stage (28-week) of fetal development observed in humans.     

Heavy meromyosin complexing filaments in the phloem of Vicia faba and Xylosma congestum

Summary Stem sections of Vicia faba L. were incubated with rabbit-muscle heavy meromyosin (HMM) and HMM complexes with phloem filaments (P-protein) were observed with the electron microscope. Treatment of sections of Vicia faba and of Xylosma congestum (Lour.) Merr. with fluorescent HMM resulted in a weak fluorescence of the phloem region. Inasmuch as HMM-binding is believed to be specific for actin-like proteins, it is proposed to classify P-protein as such.     

The Effects of the Organic Flame‐Retardant 1,2‐Dibromo‐4‐(1,2‐dibromoethyl) Cyclohexane (TBECH) on Androgen Signaling in Human Prostate Cancer Cell Lines

The effects of the organic flame retardant 1,2‐Dibromo‐4‐(1,2‐dibromoethyl) cyclohexane (TBECH) on androgen receptor target gene expression were examined in the human LNCaP prostate cancer cell line. While γ‐/δ‐TBECH alone led to a significant increase in prostate‐specific antigen (PSA) mRNA accumulation, both the α‐/‐TBECH and γ‐/δ‐TBECH mixtures repressed androgen‐inducible PSA mRNA and protein accumulation in human LNCaP cells. Thus, we hypothesize that isomeric mixtures of TBECH may act as partial agonists of the androgen receptor.     

Cell type-specific proteasomal processing of HIV-1 Gag-p24 results in an altered epitope repertoire

Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.     

Deletion and insertion mutants of the multidrug transporter

The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs. To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations. The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain. However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance. An internal deletion of the amino-terminal half, which removed residues 140-229, is also nonfunctional. Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance. Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity. These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter.     

Partial molecular genetic map of the rabbit VH chromosomal region

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.     

Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters.

The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.     

Characterization of the azidopine and vinblastine binding site of P-glycoprotein.

To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.