Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)

The mode and duration of anti-CD28 costimulation determine resistance to infection by macrophage-tropic strains of human immunodeficiency virus type 1 in vitro.

We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) in vitro. Our results confirm the observations of Levine et al. (15) that stimulation of CD4 T cells with anti-CD3/anti-CD28 antibodies coimmobilized on magnetic beads renders the cells resistant to infection by M-tropic strains of HIV-1. The resistance was strongest when the beads were left in the cultures throughout the experiment. In contrast, stimulation of CD4 T cells with the same antibodies immobilized on the surface of plastic culture dishes failed to induce resistance and resulted in high levels of p24 production. This was true even if the cells were passaged continuously on freshly coated plates. If the beads were removed after initial stimulation, p24 production increased over time and produced a result intermediate to the other forms of stimulation. For beads-in, beads-out, and one-time plate stimulated cultures, resistance to infection correlated with down-regulation of CCR5 expression at the cell surface and with increased production of beta-chemokines. However, cultures of CD4 T cells continuously passaged on anti-CD3/anti-CD28-coated plates produced large amounts of p24 despite decreased levels of CCR5 expression and increasing production of beta-chemokines. Expression of the T-cell activation markers CD25 and CD69 and production of gamma interferon further supported the differences in plate versus bead stimulation. Our results explain the apparent contradiction between the ability of anti-CD28 antibody costimulation to induce resistance to HIV infection when presented on magnetic beads and the increased ability to recover virus from the cells of HIV-positive donors who are on highly active antiretroviral therapy when cells are stimulated by anti-CD3/anti-CD28 immobilized on plastic dishes.

Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols.

BACKGROUND: The use of gene modified T lymphocytes for immunotherapy in a cancer or AIDS clinical trial requires an efficient, safe ex vivo method for modification of these cells at manufacturing scale. Since retroviruses have been shown to be a moderately effective means of stably integrating therapeutic genes into T lymphocytes, we wanted to create packaging and producer cell lines that would produce replication competent retrovirus (RCR)-free supernatants, at large scale (> 200 l), and transduce with high efficiency. METHODS: cDNA expression plasmids containing only coding sequences for gagpol or env were built and sequentially transfected into human 293 cells. Packaging and producer clones were characterized for stability, titer and RCR. A producer clone delivering chimeric immune receptors was scaled-up and supernatants used to transduce patient T lymphocytes for clinical studies. PCR and RT-PCR assays were utilized to evaluate the transmission of HERV-H sequences. Relative infectivity of producer clones pseudotyped with different envelopes was determined by transduction and RT assays. RESULTS: RCR-free, human 293 split-genome packaging lines, pseudotyped with amphotropic, xenotropic, or 10A1 envelopes, were created. A CC49 zeta producer clone was scaled-up to 5 x 54 l lots and supernatants used to safely and efficiently transduce patient T lymphocytes with minimal ex vivo manipulation. While 293 cells express HERV-H mRNA, the transmission frequency in our packaging clones was less than 1 HERV-H sequence per 5 x 10(5) proviral integrations. Additionally, 10A1 and xenotropic packaging lines had higher infectivities than the amphotropic clone. CONCLUSION: These packaging lines represent the safest configuration for the large-scale production of retroviral vectors, and are capable of producing high titer, RCR-free retroviral vector for large scale clinical use. While all three clones efficiently transduce human T lymphocytes, the 10A1 clone has the highest infectivity. These packaging cell lines will be valuable for use in human gene therapy protocols.

Production of recombinant proteins in serum-free media.

The advantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells are reviewed. The process favoured is fed-batch serum-free cell culture. This process is applicable to the majority of cell lines, is practical on the large scale, gives the lowest manufacturing cost, and can be carried out without the use of any serum.

Piecewise-planar vowel formant distributions across speakers.

A previous study [D. J. broad and H. Wakita, J. Acoust. Soc. Am. 62, 1467--1473 (1977)] showing that one female speaker's first three vowel formant frequencies clustered about a two-part piecewise-planar surface is extended to five additional speakers. For each speaker, a similar two-plane representation is found, with the rms spread of the data about the planes ranging between 69 and 103 Hz. The orientations of the planes for the different speakers are similar: The front-vowel planes make an average angle of 10 degrees to the average front-vowel plane, while the back-vowel planes make an average angle of 13 degrees to the average back-vowel plane. Nearly all these departures from the average are significant at the 99% level. The hypothesis of uniform scaling of vowel formant frequencies between speakers must therefore be rejected if it is carried strictly to three dimensions. This is also shown by the positions of the planes. The speakers do, however, group into two almost uniformly scalable subsets. Finally, the third formants of the retroflex vowels for most of the speakers are lower than would be predicted solely from exploitation of low-F3 regions of the piecewise-planar surfaces.

Escherichia coli 5'-nucleotidase: purification, properties and its release by osmotic shock.

Escherichia coli 5'-nucleotidase was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. Its molecular weight was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, by gel exclusion chromatography, and by membrane filtration; values of 66 000, 48 500, and 15 000 to 30 000, respectively, were obtained. The enzyme was completely released from bacteria by osmotic shock treatment. The apparently anomalous behaviour of 5'-nucleotidase in terms of the molecular sieving hypothesis for the release of enzymes by osmotic shock proposed by Smith & Wyatt (1974) and extended by Broad & Smith (1979) is discussed.

Piecewise--planar representation of vowel formant frequencies.

The first three formant frequencies for 778 steady-state tokens of 30 nonretroflex vowel types uttered by a female speaker are found to lie close to a piecewise-planar surface (expressed numerically as 0.634F1 +0.603F2 -- 0.485F3 -- 366 = 0, for F2 greater than 0.027F1 +1692 and 0.686F1 -- 0.528F2 -- 0.501F3 +1569 = 0, otherwise). The rms distance of the vowels from this surface is only 86 Hz. The intersection between the two planes is a line of nearly constant F2, corresponding closely to the F2 of a uniform vocal tract of the same length as our speaker's. The piecewise-planar representation also suggests a way to test the hypotheses of uniform and nonuniform formant-frequency scaling between speakers.

Clinical significance of asymmetrical vocal cord tension.

The effects of asymmetrical tension on the vibratory pattern of the vocal cords were studied in two kinds of experiments: 1) high speed motion picture photography of artificial voice production in excised canine and human larynges, and 2) computer synthesis of voice and vocal cord vibration via a theoretical model incorporating the physiological parameters required for phonation. In both approaches the asymmetrically tensed vocal cords consistently vibrated in three distinct modes which depend partly on the rest positions of the vocal cords; Type I. For rest positions at or near closure, the two cords vibrate at the same frequency with glottal closure every period, and with tense cord preceding the lax one in phase and with the line of contact moving toward the tenser cord during the closed phase. The voice produced is not hoarse; Type II. For wider rest positions glottal closure occurs irregularly, the vibrations become complex and less periodic, and the voice becomes hoarse; Type III. The glottis never closes and the vibrations become more periodic with reduced amplitude. Supplementary stroboscopic observations suggest a precedure for diagnosing tension asymmetry and the implications for surgical treatment for disorders of vocal pitch are discussed.