Nitric oxide, the biological mediator of the decade: fact or fiction?

Nitric oxide (NO), an atmospheric gas and free radical, is also an important biological mediator in animals and humans. Its enzymatic synthesis by constitutive (c) and inducible (i) isoforms of NO synthase (NOS) and its reactions with other biological molecules such as reactive oxygen species are well characterized. NO modulates pulmonary and systemic vascular tone through its vasodilator property. It has antithrombotic functions and mediates some consequences of the innate and acute inflammatory responses; cytokines and bacterial toxins induce widespread expression of iNOS associated with microvascular and haemodynamic changes in sepsis. Within the lungs, a diminution of NO production is implicated in pathological states associated with pulmonary hypertension, such as acute respiratory distress syndrome: inhaled NO is a selective pulmonary vasodilator and can improve ventilation-perfusion mismatch. However, it may have deleterious effects through modulating hypoxic pulmonary vasoconstriction. Inhibitors of NOS may be of benefit in inotrope-refractory septic shock, but toxicity of newly developed selective iNOS inhibitors have prevented clinical trials of efficacy. An expanding literature on the origins and measurement of NO in exhaled breath implicates NO as a potentially useful marker of disease activity in respiratory tract inflammation in the future. This report reviews some aspects of research into the clinical importance of nitric oxide.     

Resistance of Indonesian thin tail sheep against Fasciola gigantica and F. hepatica

Astrocytes express variable levels of MHC class II antigens depending on their activation status or exposure to certain cytokines, notably IFN-gamma. When they are induced to express higher surface densities of MHC class II molecules, astrocytes are capable of stimulating syngeneic myelin basic protein (MBP)-reactive T cells to proliferate at a modest rate and to secrete proinflammatory cytokines, such as TNF-alpha, in response to antigen. In the present investigation evidence is presented that uninduced astrocytes, whether fresh or established as clones, on which surface MHC class II molecules are expressed at a very low density, promote an antigen-dependent reduction of TCR on the surface of syngeneic T cells. Accompanying this effect on the TCR is an induction of T cell hyporeactivity and little or no production of proinflammatory cytokines. These observations suggest that the ability of the astrocyte, through varying their surface MHC class II molecules, can control the effect of antigen-induced T cell responses. In their normal state of low MHC II expression astrocytes are expected to induce no or partial, rather than full, activation of autoreactive T cells that enter the CNS, resulting in T cell hyporeactivity. Since astrocytes usually diminish the production of proinflammatory cytokines by T cells that enter the CNS, the status and control of MHC class II expression on astrocytes should be important determinants of the suppression or enhancement of in situ immune responses in the CNS.     

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.     

Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.     

Selective aortic arch perfusion using serial infusions of perflubron emulsion

With the aim of developing foetal gene therapy for cystic fibrosis, we have investigated the possibility of gene targeting to the mouse foetus with two different viral vector systems and at different times of gestation. We report here that recombinant retrovirus producing cells administered into the intra-amniotic cavity of mid- to late-gestation mouse MF1 foetuses survive in the amniotic fluid and are able to engraft to a certain extent in foetal tissues. By production of infectious virus they mediate transduction and beta-galactosidase transgene expression in neighbouring foetal tissues 24 to 72 h following injection. Retrovirus producer cells could, therefore, become a means to overcome the limitations of low retroviral titre, for in vivo foetal gene transfer. To investigate the developmental stage at which transduction of the airways and enteral systems can be obtained we also administered a highly infective first generation adenoviral vector (AdRSV beta gal) into the amniotic cavity of foetal mice between 13 to 16 days post coitus, beta-galactosidase activity was detected between 24 to 120 h after injection. The highest levels of transgene expression were generally observed between 48 to 72 h following injection of the adenoviral vector. We demonstrate that infection of the pulmonary airways is dependent on the developmental stage of the foetus and can be achieved on the 15th day of gestation.