A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120.

Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines.     

Immortalization of murine B cells in vitro with oncogene-containing retroviral vectors.

A large panel of oncogene-containing retroviral vectors has been constructed and used to infect activated murine splenic B cells to determine whether particular oncogenes are capable of directly mediating B cell immortalization. Mature B cell lines have been consistently established with some of these retroviral vectors. These B cell lines arose at a low frequency, indicating that more genetic events were required in addition to infection with the retroviral vector for immortalization to occur. All such lines were LPS-dependent and non-tumorigenic. All lines secrete IgG and express surface IgG, but not IgD or IgM. In addition, they are CD11b+ and CD23-. These cells may be derived from the CD5 "lineage" or a related B cell subset and appear to be more susceptible to immortalization than conventional B cells.     

Description of the oöcysts of three new species of coccidia (Apicomplexa: Eimeriidae) from lizards in Namibia

Three new species of coccidian were recovered from the intestinal contents and faeces of lizards in Namibia, southwest Africa. Oöcysts of Eimeria barnardi n. sp. are described from Rhoptropus barnardi (Gekkonidae) and are ellipsoidal, 24.3 × 19.9 (21–26.5 × 16–22) m; shape index (length/width) 1.22 (1.12–1.30). A micropyle and oöcyst residuum are absent but a fragmented polar granule is present. Sporocysts are subspherical, 9.2 × 8.3 (8–11 × 7.5–9) m; shape index 1.11 (1.02–1.27). Oöcysts of Eimeria pachybibroni n. sp. were found in Pachydactylus bibroni (Gekkonidae) and are ellipsoidal, 26.2 × 18.2 (21.5–28 × 16–19) m; shape index 1.44 (1.30–1.52). A micropyle and oöcyst residuum are absent but a polar granule is present. Sporocysts are subspherical, 8.9 × 8.0 (8–9.5 × 7–8.5) m; shape index 1.12 (1.03–1.20). Oöcysts of Isospora spilogaster n. sp. are reported from Mabuya spilogaster (Scincidae) and are subspherical, 27.4 × 26.0 (21.5–35 × 21–35) m; shape index 1.05 (1.00–1.13). Micropyle, oöcyst residuum and polar granules are absent. Sporocysts are ellipsoidal, 13.2 × 9.7 (10.5–15 × 9–11) m; shape index 1.36 (1.08–1.50).     

Description of the oöcysts of two new species of Eimeria (Apicomplexa: Eimeriidae) from the rough earth snake Virginia striatula (Serpentes: Colubridae) in Texas and Arkansas

Coccidian oöcysts recovered from the faeces of rough earth snakes Virginia striatula (Serpentes: Colubridae) were found to represent two previously unreported eimerians. Oöcysts of Eimeria desotoensis n. sp. were found in 5/32 (16%) of the snakes and were spherical to ellipsoidal, 18.4 × 17.2 (15–21.5 × 15–19.5) μm, with a thin, single-layered wall; their shape-index (length/width) was 1.07 (1.00–1.23). A micropyle and oöcyst residuum were absent; polar granule were present in 33% of the oöcysts. The sporocysts were ovoidal, 11.5 × 7.6 (10.5–13 × 7–8) μm, with a Stieda body; their shape-index was 1.51 (1.30–1.68). The sporocyst residuum was moderate in size and composed of a cluster of granules. Oöcysts of Eimeria hobartsmithi n. sp. were found in 2/32 (6%) of the snakes and were subspherical to ellipsoidal, 18.0 × 15.7 (16–20 × 15–17) μm, with a thin, single-layered wall; their shape-index was 1.15 (1.02–1.32). A micropyle, oöcyst residuum and polar granule were absent. The sporocysts were elongate, 13.2 × 6.3 (12–14.5 × 6–6.5) μm, with a Stieda body; their shape-index was 2.10 (1.88–2.34). A large sporocyst residuum was present in each sporocyst, often obscuring the sporozoites. In addition to the two new species, oöcysts of E. striatula Upton & McAllister, 1990 were observed in 38% of the snakes.     

Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels

Summary A new low shear stress microcarrier culture system has been developed at NASA’s Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue.     

CO2 Incorporation and 4-Hydroxy-2-Methylbenzoic Acid Formation during Anaerobic Metabolism of m-Cresol by a Methanogenic Consortium

The metabolism of m-cresol by methanogenic cultures enriched from domestic sewage sludge was investigated. In the initial studies, bromoethanesulfonic acid was used to inhibit methane production. This led to the accumulation of 4.0 ± 0.8 mol of acetate per mol of m-cresol metabolized. These results suggested that CO₂ incorporation occurred because each molecule of m-cresol contained seven carbon atoms, whereas four molecules of acetate product contained a total of eight carbon atoms. To verify this, [¹⁴C]bicarbonate was added to bromoethanesulfonic acid-inhibited cultures, and those cultures yielded [¹⁴C]acetate. Of the label recovered as acetate, 89% was found in the carboxyl position. Similar cultures fed [methyl-¹⁴C]m-cresol yielded methyl-labeled acetate. A ¹⁴C-labeled transient intermediate was detected in cultures given either m-cresol and [¹⁴C]bicarbonate or bicarbonate and [methyl-¹⁴C]m-cresol. The intermediate was identified as 4-hydroxy-2-methylbenzoic acid. In addition, another metabolite was detected and identified as 2-methylbenzoic acid. This compound appeared to be produced only sporadically, and it accumulated in the medium, suggesting that the dehydroxylation of 4-hydroxy-2-methylbenzoic acid led to an apparent dead-end product.     

Identification of Phytophthora citrophthora with Cloned DNA Probes

Two different DNA fragments, one of 2.9 kilobases and the other of 5.1 kilobases, were cloned from Phytophthora citrophthora and showed no homology with DNA from plants and other related fungi. These DNA probes hybridized with DNA from 12 different P. citrophthora isolates obtained from a variety of hosts but did not hybridize with DNA from 6 P. citrophthora isolates obtained from cacao. Southern blot analysis revealed that the probes contained repetitive DNA, and restriction fragment length polymorphisms were identified among several P. citrophthora isolates. Of the isolates tested, two major groups were observed whose genetic similarity correlated with geographical distribution. One of the DNA probes was used to detect P. citrophthora growing from infected citrus roots incubated on semiselective medium. P. citrophthora was not detected by a hybridization assay of total DNA extracted directly from infected roots.