Resistance to human immunodeficiency virus type 1 infection conferred by transduction of human peripheral blood lymphocytes with ribozyme, antisense, or polymeric trans-activation response element constructs.

Human peripheral blood lymphocytes (PBLs) were transduced with a number of recombinant retroviruses including RRz2, an LNL6-based virus with a ribozyme targeted to the human immunodeficiency virus (HIV) tat gene transcript inserted within the 3' region of the neomycin-resistance gene; RASH5, and LNHL-based virus containing an antisense sequence to the 5' leader region of HIV-1 downstream of the human cytomegalovirus promoter; and R20TAR, an LXSN-based virus with 20 tandem copies of the HIV-1 trans-activation response element sequence driven by the Moloney murine leukemia virus long terminal repeat. After G418 selection, transduced PBLs were challenged with the HIV-1 laboratory strain IIIB and a primary clinical isolate of HIV-1, 82H. Results showed that PBLs from different donors could be transduced and that this conferred resistance to HIV-1 infection. For each of the constructs, a reduction of approximately 70% in p24 antigen level relative to the corresponding control-vector-transduced PBLs was observed. Molecular analyses showed constitutive expression of all the transduced genes from the retroviral long terminal repeat, but no detectable transcript was seen from the internal human cytomegalovirus transcript was seen from the internal human cytomegalovirus promoter for the antisense construct. Transduction of, and consequent transgene expression in, PBLs did not impact on the surface expression of either CD4+/CD8+ (measured by flow cytometry) or on cell doubling time (examined by [3H]thymidine uptake). These results indicate the potential utility of these anti-HIV-1 gene therapeutic agents and show the preclinical value of this PBL assay system.

Functional and developmental studies of the peripheral arterial chemoreceptors in rat: effects of nicotine and possible relation to sudden infant death syndrome.

The drive on respiration mediated by the peripheral arterial chemoreceptors was assessed by the hyperoxic test in 3-day-old rat pups. They accounted for 22.5 +/- 8.8% during control conditions, but only for 6.9 +/- 10.0% after nicotine exposure, an effect counteracted by blockade of peripheral dopamine type 2 receptors (DA2Rs). Furthermore, nicotine reduced dopamine (DA) content and increased the expression of tyrosine hydroxylase (TH) in the carotid bodies, further suggesting that DA mediates the acute effect of nicotine on arterial chemoreceptor function. During postnatal development TH and DA2R mRNA levels in the carotid bodies decreased. Thus, nicotine from smoking may also interfere with the postnatal resetting of the oxygen sensitivity of the peripheral arterial chemoreceptors by increasing carotid body TH mRNA, as well as DA release in this period. Collectively these effects of nicotine on the peripheral arterial chemoreceptors may increase the vulnerability to hypoxic episodes and attenuate the protective chemoreflex response. These mechanisms may underlie the well-known relation between maternal smoking and sudden infant death syndrome.

Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.

Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide.

Mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play important roles in tissue inflammation and in neuroimmune interactions. Very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. Palmitoylethanolamide, found in peripheral tissues, has been proposed to behave as a local autacoid capable of downregulating mast cell activation and inflammation. A cognate N-acylamide, anandamide, the ethanolamide of arachidonic acid, occurs in brain and is a candidate endogenous agonist for the central cannabinoid receptor (CB1). As a second cannabinoid receptor (CB2) has been found in peripheral tissues, the possible presence of CB2 receptors on mast cells and their interaction with N-acylamides was investigated. Here we report that mast cells express both the gene and a functional CB2 receptor protein with negative regulatory effects on mast cell activation. Although both palmitoylethanolamide and anandamide bind to the CB2 receptor, only the former downmodulates mast cell activation in vitro. Further, the functional effect of palmitoylethanolamide, as well as that of the active cannabinoids, was efficiently antagonized by anandamide. The results suggest that (i) peripheral cannabinoid CB2 receptors control, upon agonist binding, mast cell activation and therefore inflammation; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for the CB2 receptor on mast cells; (iii) modulatory activities on mast cells exerted by the naturally occurring molecule strengthen a proposed autacoid local inflammation antagonism (ALIA) mechanism; and (iv) palmitoylethanolamide and its derivatives may provide antiinflammatory therapeutic strategies specifically targeted to mast cells ("ALIAmides").

Rapid alteration of thalamocortical axon morphology follows peripheral damage in the neonatal rat.

The effect of day of birth (postnatal day 0; P0) infraorbital nerve section on the morphology of individual thalamocortical axons in rat somatosensory cortex was examined on P3. Thalamic fibers were labeled in fixed brains with the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, and individual photo-converted thalamocortical fibers were reconstructed. In normal animals on P3, axon arbor terminal formation within layer IV has commenced and terminal arbor width is comparable to that of a cortical "barrel." After infraorbital nerve section, the average width of thalamocortical terminal arbors is significantly greater than is the average arbor width of normal rats of the same age; however, neither the number of branches per terminal arbor nor total arbor length differs between groups. These observations suggest that the role of the periphery in guiding terminal arbor formation is exerted both very rapidly and at the level of the single thalamic axon. Further, these results indicate a close association between individual axon terminal arbor morphology and pattern formation in the rat somatosensory cortex.