Three strategies have been designed to concentrate infectious retroviral vectors from the supernatants of human- (HT1080) and murine- (NIH 3T3) based packaging cells. Streptavidin-conjugated paramagnetic particles in conjunction with (i) antibodies directed against murine fibronectin, (ii) biotinylated lectins, or (iii) biotin-modified packaging cell-surface proteins allow affinity-mediated magnetic concentration of retroviral vectors. Retroviral titers (assayed by colony formation of human myeloid K562 cells) are increased by 1-4 x 10(3)-fold after volume reductions of only 125-fold. Using these procedures, preparations of 5 x 10(8) cfu/ml are routinely made from relatively low-titer (2-5 x 10(5) cfu/ml) starting material. High-titer (paramagnetic) retroviral vector preparations can be used for magnetic field-dependent retroviral infection in vitro. Magnetic field-dependent localization such as this may enable the in vivo administration of formulations that concentrate retroviral infection to the required target tissues and organs.
Antigen-specific T lymphocytes are attractive as potential anticancer agents. The generation of large numbers of antigen-specific T cells is possible through the use of gene therapy to express targeting receptors on the T lymphocyte. Activated T lymphocytes were transduced to express carcino-embryonic antigen or neural cell adhesion molecule targeted CD3zeta chimeric immune receptors. The chimeric receptors were expressed as homodimers and also as heterodimers with the native CD3zeta. T lymphocyte populations were expanded in the absence of selection for the modified cells and were shown to produce cytokines when cultured in the presence of immobilized purified protein antigen. These lymphocytes also responded by cytokine production and cytolytic activity when challenged with tumor-cell lines expressing the antigen recognized by the chimeric immune receptor. The cytolytic activity appears to be largely perforin mediated. Furthermore, soluble carcino-embryonic antigen did not interfere with the functional activity of the carcino-embryonic antigen-targeted lymphocytes. Long-term (5-day) stimulation of the modified lymphocytes by protein antigen resulted in reduced viability similar to that induced by anti-CD3 antibodies alone. Viability was improved by a costimulatory signal indicating that such signals may be vital in the maintenance of long-term functional activity of receptor modified T lymphocytes.
Ross River virus has been incriminated in the etiology of many sporadic and epidemic cases of polyarthritis in Australia and the Pacific. Both synovium and synovial exudate fluid recovered from the knee of an epidemic polyarthritis patient showed a predominantly mononuclear leucocyte infiltrate. Infectious virus could not be recovered from the synovial exudate. Functional natural killer cells were detected in the synovial fluid. Their level of cytotoxic activity was similar to that detected in the peripheral circulation.
To evaluate the role of perinatal transmission in the spread of hepatitis C virus (HCV), we screened a cohort of pregnant intravenous drug using (IVDU) women for HCV antibody detection; where seropositive HCV RNA detection by polymerase chain reaction (PCR) was found we followed the infants longitudinally for HCV antibody and HCV RNA. Serum prevalence for HCV for this population was 80% with HCV RNA detected in 50%. Recruitment and follow-up over a 3-year period of a cohort of 83 seropositive women, their 91 newborns and 16 siblings of newborns, showed that there had been a 3% perinatal transmission rate with 1 sibling also infected. These positive cases were defined as transient in 1 case (HCV RNA positive by PCR at 1 month, but seronegative and HCV RNA negative at 10 months of age), 2 unevaluable (HCV RNA positive at 2 months of age, but patients lost to follow-up), and 1 chronic infection in a child at 34 months (positive HCV RNA and seropositive 34-month sibling). Maternal HCV RNA levels for those with infected infants was a mean 40-fold greater than those whose babies were uninfected, although this did not reach statistical significance. Of the remaining infants, the majority (93%) had lost passively acquired maternal antibodies by 9 months of age and all by 12 months. Of 18 women who were HCV seropositive and breast feeding (66% of whom were HCV RNA positive in their sera), none had detectable HCV RNA in breast milk. Hence we conclude that transmission of HCV from mother to infant appears to be of low frequency and positivity appears to correlate with maternal circulating viral load.
We have studied the effect of NE 19550 (olvanil, N-(4-hydroxy-3-methoxyphenyl) methyl-9Z-octadecenamide), a capsaicin analogue with approximately equipotent antinociceptive activity in vivo compared with capsaicin, on nociceptive responses recorded from spinal dorsal horn neurones in vivo and from a spinal ventral root in vitro. In adult rats anaesthetized with halothane, antinociceptive doses of olvanil (20-40 mumol/kg, s.c.) reduced C-fibre responses evoked in wide dynamic range, lumbar dorsal horn neurones, by peripheral transcutaneous electrical stimulation. Intradermal injection of olvanil, localized to a discrete region of the peripheral receptive field, did not activate C-fibres nor change C-fibre evoked activation of dorsal horn neurones. Spinal intrathecal administration of olvanil attenuated C-fibre evoked responses and, at the highest concentration, significantly reduced A beta-fibre evoked activity. In the neonatal rat spinal cord/tail preparation maintained in vitro, superfusion of the cord with olvanil (500 nM-5 microM) did not evoke a depolarization but responses to peripheral noxious stimulation were attenuated. In a similar in vitro preparation of the neonatal rat spinal cord, the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was measured in spinal cord superfusates. Capsaicin (2-10 microM) evoked a large release of CGRP-LI but olvanil (2-10 microM) produced only a small or undetectable release. Following the administration of each substance, however, the release of CGRP-LI evoked by a depolarizing potassium stimulus was significantly attenuated. These data indicate that C-fibre input to the dorsal horn was attenuated by acute systemic doses of olvanil that were antinociceptive in behavioural tests. This effect was unlikely to be due to impairment of C-fibre function by a peripheral locus of action but was more consistent with an action in the spinal cord in which the reduced release of a neurotransmitter substance from afferent nerve terminals may play a prominent role.
For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of
SUMMARY1. The sequence of events involved in degeneration of sympathetic nerve terminals has been studied in vitro in the expansor secundariorum muscle of the chicken.2. The muscle underwent a degeneration contraction due to abrupt release of noradrenaline stores from the nerves about 18 hr after isolation. 6. When the muscle was left in situ after denervation, the degeneration contraction occurred between 2 and 3 days later depending on the length of the distal portion of nerve. Cutting the nerve supply 5 cm closer to the muscle in vivo hastened the loss of transmission and the onset of the degeneration contraction by 24-48 hr.7. Supersensitivity to noradrenaline occurred soon after the degeneration contraction but the full development of a sevenfold supersensitivity to noradrenaline, equivalent to that produced by cocaine, took up to 48 hr. No further increase in sensitivity to noradrenaline could be detected in vitro after chronic denervation in vivo for up to 30 days.L. B. GEFFEN AND CHRIS C. HUGHES 8. It is concluded that the degeneration contraction and failure of transmission was precipitated by an abrupt increase in the Ca2+ permeability of the axon causing discharge of transmitter from vesicles. This was probably the result of depletion of essential factors normally transported somatofugally in the axon at 1-2 mm/hr. The supersensitivity to noradrenaline that develops subsequently appears to be due to a loss of the uptake mechanism of the nerve terminals.
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