Development of a magnetron-enhanced plasma process for tungstenetchback with response-surface methodology

A two-step etchback process to form tungsten plugs in submicron contacts and vias has been developed. the process uses an Applied Materials Inc., P5000 WCVD magnetron-enhanced, single-wafer system with an experimental design and response-surface methodology. Tungsten is first etched with an Ar/SF₆ mixture until excited N₂ molecules from the underlying TiN adhesion layer are detected in the plasma. Residual TiN is then etched for a fixed time with an Ar/Cl ₂ plasma. Both steps employ a rotating 0.5-Hz magnetic field. Although the use of the magnetic field has no pronounced effect on the etch rate of either film, it provides broad regions of high etch uniformity. In addition, the DC-bias voltage measured as part of the TiN study decreases with increasing magnetic field strength without reducing the etch rate of the film     

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.     

Analysis of cytokine mRNA profiles in the lungs of Pneumocystis carinii-infected mice

Severe combined immunodeficient (scid) mice lack functional CD4+ lymphocytes, and therefore develop life-threatening Pneumocystis carinii infection. However, when scid mice are immunologically reconstituted with spleen cells, including CD4+ cells, a protective inflammatory response is mounted against the organism. To determine whether these lymphocytes induce elevated cytokine mRNA levels in response to P. carinii infection, steady-state levels of cytokine mRNAs were measured in the lungs of both reconstituted and unaltered scid mice. Despite significant numbers of organisms and the presence of functional alveolar macrophages in the lungs of 8- and 10-wk-old scid mice, there was neither evidence of pulmonary inflammation, nor increased proinflammatory cytokine expression. However, when 8-wk-old scid mice were immunologically reconstituted, signs of intense, focal pulmonary inflammation were observed, and levels of interleukin (IL)-1alpha, IL-1beta, IL-3, IL-6, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF)-alpha, and TNF-beta mRNAs were all significantly elevated. Cytokine expression was increased at day 10 post-reconstitution (PR), maximal at day 12 PR, and returned to baseline by day 22 PR. In situ hybridization demonstrated that at day 12 PR, increased IL-1beta and TNF-alpha expression was localized to sites of intense inflammation and focal P. carinii colonization. Many of the cells expressing high levels of IL-1beta and TNF-alpha in these regions were in direct contact with organisms, or contained degraded organisms within their cytoplasm. Thus, even though functional macrophages are present in scid mice, CD4+ T cells are required for proinflammatory cytokine expression, which is associated with the generation of a protective inflammatory response at sites of P. carinii infection.     

Regenerating sciatic nerve does not utilize circulating cholesterol

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.