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Highly selective nanoscale etching of a low-dielectric constant (low-k) organosilicate (SiCOH) layer using a mask pattern of chemical-vapor-deposited (CVD) amorphous carbon layer (ACL) was carried out in CF4/C4F8/Ar dual-frequency superimposed capacitively-coupled plasmas. The etching characteristics of the SiCOH layers, such as the etch rate, etch selectivity, critical dimension (CD), and line edge roughness (LER) during the plasma etching, were investigated by varying the C4F8 flow rate. The C4F8 gas flow rate primarily was found to control the degree of polymerization and to cause variations in the selectivity, CD and LER of the patterned SiCOH layer. Process windows for ultra-high etch selectivity of the SiCOH layer to the CVD ACL are formed due to the disproportionate degrees of polymerization on the SiCOH and the ACL surfaces.

For fabrication of a multilevel resist (MLR) structure with silicon nitride (Si3N4) and amorphous carbon (a-C) layers, highly selective etching of the Si3N4 layer using a chemical vapor-deposited (CVD) a-C etch mask was investigated by varying the following process parameters in CH2F2/H2/Ar plasmas: etch gas flow ratio, high-frequency source power (PHF), and low-frequency source power (PLF) in a dual-frequency superimposed capacitively coupled plasma etcher. The results of etching the ArF photoresist/bottom antireflective coating/SiOx/CVD a-C/Si3N4 MLR structure showed the possibility of obtaining an infinitely high selective etch process for the Si3N4 layer using a thin CVD a-C etch mask for high aspect-ratio pattern formation. The CH2F2/H2 gas flow ratio was found to play a critical role in determining the process window for infinite Si3N4/CVD a-C etch selectivity, due to the change in the degree of polymerization on Si3N4 and CVD a-C surfaces.

Currently, extreme ultraviolet lithography (EUVL) is being investigated for next generation lithography. Among the core EUVL technologies, mask fabrication is of considerable importance due to the use of new reflective optics with a completely different configuration than those of conventional photolithography. This study investigated the etching properties of indium tin oxide (ITO) binary mask materials for EUVL, such as ITO (absorber layer), Ru (capping/etch-stop layer), and a Mo–Si multilayer (reflective layer), by varying the Cl2/Ar gas flow ratio, dc self-bias voltage (Vdc), and etch time in inductively coupled plasmas. The ITO absorber layer needs to be etched with no loss in the Ru layer on the Mo–Si multilayer for fabrication of the EUVL ITO binary mask structure proposed here. The ITO layer could be etched with an infinitely high etch selectivity over the Ru etch-stop layer in Cl2/Ar plasma even with a very high overetch time.

The process window for infinitely high etch selectivity of silicon oxynitride (SiON) layers to extreme ultraviolet (EUV) resist and the variation in line edge roughness (LER) of etched EUV resist were investigated in a CH2F2∕H2∕Ar dual-frequency superimposed capacitively coupled plasma under various process parameters including the gas flow ratio and low-frequency source power (PLF). The CH2F2∕H2 gas flow ratio was found to play a critical role in determining the process window for infinite selectivity of the SiON/EUV resist due to the change in the degree of polymerization. The preferential chemical reaction of hydrogen and carbon species in the hydrofluorocarbon (CHxFy) layer with nitrogen on the SiON surface led to the formation of HCN etch by-products, resulting in a thinner steady-state CHxFy layer. During continuous SiON etching, the thinner steady-state CHxFy layer was due to enhanced SiF4 formation, while the CHxFy layer was deposited on the EUV resist surface. In addition, the critical dimension ...

A multilevel resist (MLR) structure can be fabricated based on a very thin amorphous carbon (a-C) layer (≅80nm) and Si3N4 hard-mask layer (≅300nm). The authors investigated the selective etching of the Si3N4 layer using a physical-vapor-deposited (PVD) a-C mask in a dual-frequency superimposed capacitively coupled plasma etcher by varying the process parameters in the CH2F2∕H2∕Ar plasmas, viz., the etch gas flow ratio, high-frequency source power (PHF), and low-frequency source power (PLF). They found that under certain etch conditions they obtain infinitely high etch selectivities of the Si3N4 layers to the PVD a-C on both the blanket and patterned wafers. The etch gas flow ratio played a critical role in determining the process window for infinitely high Si3N4∕PVD a-C etch selectivity because of the change in the degree of polymerization. The etch results of a patterned ArF photoresisit/bottom antireflective coating/SiOx∕PVD a-C∕Si3N4 MLR structure supported the idea of using a very thin PVD a-C layer a...

For gaplling of nano-scale shallow trench isolation (STI) with no voids in high-density plasma chemical vapor deposition (HDP-CVD), an etch gas containing NF3 in the continuous deposition/etch or cyclic deposition-etch-deposition (DED) processes has been adopted and in turn its use has led to uorine incorporation. Since F incorporation observed during the gapll process is known to a ect the post-process after gapll, a control and an understanding of uorine incorporation in the etch step is required. Fluorine incorporation in the HDP-CVD oxide was investigated by varying the NF3/O2 and the NF3/Ar plasma conditions during the etch step in the reactive ion etching (RIE) and the chemical dry etching (CDE) modes. As the ow ratio of NF3 gas was increased, the uorine incorporation increased. As a result of the increased F incorporation with increasing NF3 ow ratio, wet etch rates of plasma-etched HDP oxides increased.

For the fabrication of a multilevel resist (MLR) based on a very thin, physical-vapor-deposited (PVD) amorphous carbon (a-C) layer, the etching characteristics of the PVD a-C layer with a SiOx hard mask were investigated in a dual-frequency superimposed capacitively coupled plasma etcher by varying the following process parameters in O2∕N2∕Ar plasmas: high-frequency/low-frequency combination (fHF∕fLF), HF/LF power ratio (PHF∕PLF), and O2 and N2 flow rates. The very thin nature of the a-C layer helps to keep the aspect ratio of the etched features low. The etch rate of the PVD a-C layer increased with decreasing fHF∕fLF combination and increasing PLF and was initially increased but then decreased with increasing N2 flow rate in O2∕N2∕Ar plasmas. The application of a 30nm PVD a-C layer in the MLR structure of ArF PR∕BARC∕SiOx∕PVD a-C∕TEOS oxide supported the possibility of using a very thin PVD a-C layer as an etch-mask layer for the TEOS-oxide layer.

The tumor necrosis factor receptor (TNFR) superfamily consists of approximately 10 characterized members of human proteins. We have identified a new member of the TNFR superfamily, TR2, from a search of an expressed sequence tag data base. cDNA cloning and Northern blot hybridization demonstrated multiple mRNA species, of which a 1.7-kilobase form was most abundant. However, TR2 is encoded by a single gene which, maps to chromosome 1p36.22-36.3, in the same region as several other members of the TNFR superfamily. The most abundant TR2 open reading frame encodes a 283-amino acid single transmembrane protein with a 36-residue signal sequence, two perfect and two imperfect TNFR-like cysteine-rich domains, and a short cytoplasmic tail with some similarity to 4-1BB and CD40. TR2 mRNA is expressed in multiple human tissues and cell lines and shows a constitutive and relatively high expression in peripheral blood T cells, B cells, and monocytes. A TR2-Fc fusion protein inhibited a mixed lymphocyte reaction-mediated proliferation suggesting that the receptor and/or its ligand play a role in T cell stimulation.

K46J B lymphomas express a T cell costimulatory activity that is not inhibited by CTLA-4Ig, anti-B7-1, anti-B7-2, anti-intercellular adhesion molecule 1 or antibodies to heat stable antigen. In this paper we report that this costimulatory activity is mediated at least in part by 4-1BB ligand, a member of the tumor necrosis factor (TNF) gene family that binds to 4-1BB, a T cell activation antigen with homology to the TNF/nerve growth factor receptor family. A fusion protein between 4-1BB and alkaline phosphatase (4-1BB-AP) blocks T cell activation by K46J lymphomas in both an antigen-specific system and with polyclonally (anti-CD3) activated T cells. 4-1BB-AP also blocks antigen presentation by normal spleen cells. When the antigen-presenting cells express B7 molecules as well as 4-1BB ligand, we find that B7 molecules and 4-1BB-AP both contribute to T cell activation. These data suggest that 4-1BB ligand plays an important role in costimulation of IL-2 production and proliferation by T cells. The B lymphoma M12 expresses low levels of 4-1BB-L but can be induced to express higher levels by treatment of the B cells with cAMP, which also induces B7-1 and B7-2 in these cells. Thus cAMP appears to coordinately induce several costimulatory molecules on B cells.

Summary Autoimmune diseases are characterized by the body's ability to mount immune attacks on self. This results from recognition of self-proteins and leads to organ damage due to increased production of pathogenic inflammatory molecules and autoantibodies. Over the years, several new potential therapeutic targets have been identified in autoimmune diseases, notable among which are members of the tumour necrosis factor (TNF) superfamily. Here, we review the evidence that certain key members of this superfamily can augment/suppress autoimmune diseases.

Human interleukin-9 (IL-9) was originally identified and cloned based on its stimulatory effect on proliferation of human myeloid cell line, M07e. IL-9 synergized with Steel factor, the ligand for the c-kit product, to stimulate M07e cell proliferation. To investigate potential mechanisms for this, IL-9 was assessed for effects on protein tyrosine kinase activities in M07e cells by immunoblotting with anti- phosphotyrosine monoclonal antibody; results were compared with those of Steel factor alone and in combination with IL-9, and those of 12–0- tetradecanoyl phorbol-13-acetate (TPA). Recombinant human IL-9 (10 ng/mL) rapidly and transiently induced or enhanced at least four tyrosine phosphorylated protein bands with molecular weights of 105, 97, 85, and 81 Kd. This tyrosine phosphorylation pattern was different from that generated by recombinant murine Steel factor or TPA stimulation and the combination of IL-9 and Steel factor did not change the IL-9-induced pattern. IL-9-induced tyrosine phosphorylated bands were completely blocked by treatment of IL-9 with anti-IL-9 antibody under conditions that also neutralized the synergistic effect of IL-9 with Steel factor on M07e cell proliferation. Genistein, a tyrosine kinase inhibitor, blocked phosphorylation of IL-9 and Steel factor- induced bands. Unlike Steel factor or TPA, IL-9 did not appear to stimulate phosphorylation of 42-Kd mitogen-activated protein (MAP) kinase or Raf-1, or enhance MAP kinase activity. MAP kinase and Raf-1 are serine/threonine kinases that are phosphorylated and activated by many growth factors and by agonists for protein kinase C. While the combination of IL-9 plus SLF did not appear to induce phosphorylation of new bands not already seen with either IL-9 or SLF alone, or enhance the phosphorylation of those bands seen with either cytokine alone, the results suggest that IL-9 activates specific and unique signal transduction pathways.

Eighteen variants recovered from specific locus mutation rate experiments involving the mutagen chlorambucil were subjected to several genetic and molecular analyses. Most mutations were found to be homozygous lethal. Because lethality is often presumptive evidence for multilocus-deletion events, 10 mutations were analyzed by Southern blot analysis with probes at, or closely linked to, several of the specific locus test markers, namely, albino (c), brown (b), and dilute (d). All eight mutations (two c; three b; two d; and one dilute-short ear [Df(d se)]) that arose in post-spermatogonial germ cells were deleted for DNA sequences. No evidence for deletion of two d-se region probes was obtained for the remaining two d mutations that arose in stem-cell spermatogonia. Six of the primary mutants also produced low litter sizes ("semisterility"). Karyotypic analysis has, to date, confirmed the presence of reciprocal translocations in four of the six. The high frequency of deletions and translocations among the mutations induced in post-spermatogonial stages by chlorambucil, combined with its overall high efficiency in inducing mutations in these stages, should make chlorambucil mutagenesis useful for generating experimentally valuable germ-line deletions throughout the mouse genome.

Genomic clones encompassing the entire coding region of the mouse lymphocyte pore-forming protein gene (Pfp) have been isolated and used to determine its intron-exon organization. In contrast to C9, Pfp has a simple structure, consisting of only three exons (two of which encode polypeptide), a large 5' intron, and a single, smaller intron that is situated approximately one-third of the way through the protein-coding portions of the gene. The regions encoding the homologous domains of PFP and C9 are encoded on exons 7, 8, 9, and 10 of C9, but form only approximately half of the open reading frame of exon III in Pfp. Although encoding polypeptides with related functions, the two genes possess such sharply contrasting structures as to suggest that their analogous regions may have risen independently, by a process of convergent evolution. Using a panel of somatic cell hybrid cell lines, Pfp has been mapped to chromosome 10.

Chemotactic cytokines, referred to as chemokines, play an important role in leukocyte trafficking. The circulating levels of chemokines have been shown to increase in inflammatory processes including obesity-related pathologies (e.g. atherosclerosis and diabetes). However, little is currently known about the relationship between chemokines and human obesity. In the present study, we investigated the circulating levels of selected chemokines (monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), leukotactin-1, interleukin-8 (IL-8)) and the association between the chemokine levels and obesity-related parameters: body mass index (BMI), waist circumference, fasting glucose and insulin levels, lipids profile, and the level of C-reactive protein (CRP).A total of 100 subjects, 50 obese (BMIor=25 kg/m2) and 50 who were not obese (BMI25 kg/m2) participated in the present study. The levels of chemokines and CRP were measured in a fasting state serum by sandwich enzyme-linked immunosorbent assay. Total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, glucose, and insulin levels were measured by enzymatic analysis and immunoassay.The circulating levels of MCP-1 and IL-8 in the serum were significantly (P0.05) higher in obese subjects (BMI30 kg/m2) compared with those of nonobese controls (BMI25 kg/m2). The levels of CRP were positively correlated with BMI (P0.001) or waist circumference (P0.0001). The levels of MCP-1 and IL-8 were positively related to BMI (MCP-1, P0.02; IL-8, P0.01) and/or waist circumference (MCP-1, P0.009; IL-8, P0.03). The levels of MCP-1 were positively related to the levels of CRP (P0.007) or interleukin-6 (IL-6) (P0.0001), and negatively related to the levels of HDL-cholesterol (P0.01). Homeostasis model assessment (HOMA) score was positively related to the levels of MCP-1 (P0.02) or IL-8 (P0.03) in obese subject.Our data demonstrated that the circulating levels of MCP-1 and IL-8 are related to obesity-related parameters such as BMI, waist circumference, CRP, IL-6, HOMA and HDL-cholesterol. These findings suggest that the circulating MCP-1 and/or IL-8 may be a potential candidate linking obesity with obesity-related metabolic complications such as atherosclerosis and diabetes.

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is induced on CD4+ and CD8+ T cells upon engagement of the T cell receptor (TCR)/ CD3 complex with the antigen bound to MHC. 4-1BB plays an important role in transmitting costimulatory signal during T cell activation. However, 4-1BB-mediatded signal transduction pathways have remained elusive. We conducted the yeast two-hybrid screening to identify intracellular signaling molecules that associate with 4-1BB. A novel leucine-rich repeat (LRR)-containing protein, herein named LRR-1, was found to specifically interact with the cytoplasmic domain of 4-1BB. Overexpression of LRR-1 suppressed the activation of NF-KB induced by 4-1BB or TNF receptor-associated factor (TRAF) 2. In addition, LRR-1 down-regulated JNK1 activity was induced by 4-1BB. These results indicate that LRR-1 negatively regulates the 4-1BB-mediated signaling cascades which result in the activation of NF-kappaB and JNK1.

The2',5'-oligoadenylate-dependent RNase L gene functions in the interferon-inducible RNA decay pathway known as the 2-5A system. The purpose of this study was to determine whether the absence of this gene affects the pathogenesis of herpes simplex virus type 1 (HSV-1) ocular infection in the mouse.HSV-1 (strain McKrae) was applied bilaterally to unscarified corneas of RNase L-null mice and congenic controls. To evaluate the severity of herpetic keratitis, slit lamp examinations (SLE) were performed every other day for 14 days. To study corneal histology and apoptosis, HSV-1-inoculated RNase-L-null and congenic control mice, as well as mock-inoculated mice (apoptosis negative control), were killed at 6 and 18 hours postinoculation (PI). Uninoculated mice that underwent corneal scarification (apoptosis positive control) were killed 2 hours after scarification. Eyes were dissected and the corneas processed for light and transmission electron microscopy and the TUNEL assay.In comparison with the congenic control mice, RNase L-null mice showed significantly more severe herpetic keratitis (PI day 8, SLE score, mean +/- SEM: 3.27 +/- 0.10 vs. 2.34 +/- 0.06; P:0.001) and significantly higher mortality (PI day 14, 70% vs. 20%; P:0.001). Few apoptotic cells were seen in HSV-1-infected RNase L-null mice, although DNA fragmentation consistent with apoptosis was detected in the corneas of congenic control mice 6 and 18 hours after HSV-1 inoculation and in uninfected mice with scarified corneas. Signs of apoptosis were not present in the mock-infected corneas. Electron microscopic evidence of keratocytic apoptosis was detected only in the uninfected scarified corneas and the HSV-1-infected congenic control corneas.The increased severity of ocular disease and increased mortality in the RNase L-null mice provides evidence, for the first time, that the 2-5A system contributes to protection during ocular herpetic infection. The reduced frequency of apoptosis in these mice suggests that one possible mechanism for this protective effect could be the induction of apoptosis in corneal cells as a means of reducing the spread of infectious virus.

Two new members of human beta-chemokine cDNA were isolated based on structural and functional similarities to human leukotactin-1. One of these clones was identical to the previously isolated human beta-chemokine, CKbeta8, whereas the other is a splicing variant of CKbeta8, therefore named CKbeta8-1. CKbeta8 was short in 51 nucleotides (17 amino acids) compared with CKbeta8-1. The mature proteins of CKbeta8-1 and CKbeta8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CKbeta8-1 and CKbeta8 were potent agonists at CCR1. These chemokines chemoattracted neutrophils, monocytes, and lymphocytes. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. To our knowledge, this is the first example that an alternative splicing produces two active beta-chemokines from a single gene.

Chemokines refer to a rapidly expanding family of small cytokines whose primary function is recruitment of leukocytes to inflammatory sites. These are known to bind to seven-transmembrane-domain containing receptors. A cDNA clone, CHEMR1, resembling the typical G protein-coupled receptor, was isolated from a mouse cytotoxic T-lymphocyte (CTL) library. Northern blot analysis in mouse cell lines suggests that its expression is found in a variety of cells, including T cells, B cells, and macrophages. The CHEMR1 gene Scya3r2 is a single-copy gene whose open reading frame may be in a single exon and maps to the distal region of mouse Chr 9 where the mouse macrophage inflammatory protein-1alpha (MIP-1alpha) receptor gene Scya3r and two related C-C chemokine receptor-like genes reside. Amino acid sequence comparison shows that CHEMR1 is 84% identical to human CCR-4, indicating that CHEMR1 is likely to be a mouse CCR-4. Binding assays using 125I-labeled C-C chemokines in mammalian cells indicated that CHEMR1 did not bind MIP-1alpha, RANTES, or MIP-1beta, whereas CCR-1 binds MIP-1alpha and RANTES. Our result is different from the reported properties of human CCR-4. This suggests that CHEMR1 may be a receptor for unidentified C-C chemokine or a low-affinity receptor for MIP-1alpha.

Pmel 17 is preferentially expressed in pigment cells in a manner suggestive of involvement in melanin biosynthesis. The gene is identical to the silver (si) pigmentation locus in mice. We now produced a recombinant glutathione-S-transferase-human Pmel 17 infusion protein and raised polyclonal antibodies against it to confirm the ultrastructural location and presumed site of action predicted by the deduced primary structure of Pmel 17/silver, and to authenticate the specificity of the DHICA converting function as inherent to the silver-locus protein. Full-length Pmel 17 cDNA also produced in insect cells in a baculovirus expression vector to ensure that activity did not originate from a co-precipitated protein. Natural hPmel 17 from human melanoma cells has an approximate molecular size of 100 kDa. By immunoperoxidase electron microscopic cytochemistry, the antigen was localized to the limiting membranes of premelanosomes and presumed premelanogenic cytosolic vesicles and, to a minor extent, in the premelanosomal matrix. In an in vitro assay, both the natural and the recombinant Pmel 17 accelerated the conversion of DHICA to melanin. This activity was inhibited by the anti-Pmel 17 polyclonal antibodies, indicating that the acceleration of DHICA conversion by natural protein is genuine and cannot be due to contaminating complexed proteins. We suggest that in situ Pmel 17/silver is a component of a postulated premelanosomal/melanosomal complex of membrane-bound melanogenic oxidoreductive enzymes and cofactors, in analogy to the electron transfer chain in mitochondria.

The silver mutation in mice causes progressive graying of hair due to the loss of functional follicular melanocytes. Recently the silver locus gene (called Pmel 17) has been cloned; its encoded product shares homology with a chick melanosomal matrix protein and a bovine retinal pigment epithelial protein. Although the sequence of the silver gene and the correlation of its expression with pigment production have been reported, its function in melanogenesis is still unknown. In an effort to characterize that function, we have synthesized the predicted carboxyl-terminal peptide of the mouse Pmel 17 protein and generated a rabbit polyclonal antibody (alpha PEP13) to it; that antibody recognized the silver protein specifically. The immunoaffinity-purified silver protein lacked all of the known melanogenic catalytic activities which other tyrosinase-related proteins (TRP) have, nor did it appear to modulate any of those TRP activities. Metabolic labeling experiments demonstrated that the silver protein disappears in vivo within a few hours, indicating that it is rapidly degraded, or quickly processed to lose its carboxyl terminus. Cross-reactivity experiments showed that a recently reported anti-melanosomal matrix protein antibody (alpha MX) also recognizes the silver protein, although at a different epitope from that of alpha PEP13. Using Western immunoblotting, we analyzed subcellular fractions isolated from B16 F10 melanoma cells and found that the silver protein was rich in the melanosome fraction but was absent from coated vesicles which deliver TRPs to melanosomes. These results suggest that the silver locus product is a melanosomal matrix protein which may contribute to melanogenesis as a structural protein, although the possibility remains that it also has a novel catalytic function in melanogenesis.

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

Sequence analysis of the tyrosinase coding region from an individual with tyrosinase-negative oculocutaneous albinism revealed that the patient was a compound heterozygote. One allele carried a C--> A single-base substitution in codon 355 of exon 3, and the other carried a two-nucleotide deletion in exon 1. The nucleotide substitution caused a putative amino acid change from threonine (ACA) to lysine (AAA), abolishing a signal for N-glycosylation. The two base-pair deletion caused a frameshift, creating a putative premature termination signal at codon 226. The melanocytes from the proband and her affected brother were amelanotic and devoid of measurable tyrosinase activity. Moreover, gel electrophoretic analysis of the immunoprecipitated proband tyrosinase showed that the protein was not processed to the mature glycosylated form, confirming the predicted consequence of the amino acid change. The two-base deletion on the homologous allele was detected only by sequencing genomic DNA. The transcript of this allele was not represented in the cDNA library and could not be detected by PCR mRNA, and the putative truncated protein (approximately 25 kDa) was not present in immunoprecipitates, suggesting that the allele with the missense mutation may be preferentially expressed.

We have previously reported that cytotoxic lymphocytes, when exposed to sensitive target cells, temporarily lose their lytic potential. The mechanism leading to this loss of lytic activity is still unknown but it is reversible and the lytic potency can be recovered when the effector cells are incubated with interleukin-2 (IL-2) for 12-14 hr. In this study, we have investigated the regulation of RNA coding for perforin and for two serine proteases, HSP1 and HSP2, in cytotoxic lymphocytes exposed to sensitive targets. Perforin and the two serine proteases are contained in granules of major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxic lymphocytes, but their exact role in the lytic mechanism is still debated. Here we used four different human cytotoxic lymphocytes (CTL) as effector cells: an MHC-restricted CTL (SG-CTL), a non-MHC-restricted CTL (IE6), a natural killer (NK)-like cell line (3.3) and lymphokine-activated killer (LAK) cells. In all effector cells we observed a rapid loss of perforin and of serine protease RNAs within 5 min following the addition of sensitive targets. The effector cells recovered the RNA messages as early as 30 min, although the kinetics of recovery was faster with CTL than with NK-like or LAK effector cells. When we exposed the effector cells to resistant targets we did not detect any loss of perforin or serine protease RNAs. Incubation of the effector cells with cycloheximide, prior to the addition of sensitive targets, did not block message loss, indicating that de novo protein synthesis was not required in this process. Cycloheximide treatment, however, inhibited the recovery of perforin and serine protease RNAs. Taken together, our results indicate that the target-mediated loss of lytic activity in cytotoxic lymphocytes may be a consequence of the down-regulation of perforin or of serine protease transcripts, or both.

Purified recombinant (r) macrophage inflammatory proteins (MIPs) 1 alpha, 1 beta, and 2 were assessed for effects on murine (mu) and human (hu) marrow colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies. Recombinant MIP-1 alpha, -1 beta, and -2 enhanced muCFU-GM colonies above that stimulated with 10 to 100 U natural mu macrophage-colony-stimulating factor (M-CSF) or rmuGM-CSF, with enhancement seen on huCFU-GM colony formation stimulated with suboptimal rhuM-CSF or rhuGM-CSF; effects were neutralized by respective MIP-specific antibodies. Macrophage inflammatory proteins had no effects on mu or huBFU-E colonies stimulated with erythropoietin (Epo). However, natural MIP-1 and rMIP-1 alpha, but not rMIP-1 beta or -2, suppressed muCFU-GM stimulated with pokeweed mitogen spleen-conditioned medium (PWMSCM), huCFU-GM stimulated with optimal rhuGM-CSF plus rhu interleukin-3 (IL-3), muBFU-E and multipotential progenitors (CFU-GEMM) stimulated with Epo plus PWMSCM, and huBFU-E and CFU-GEMM stimulated with Epo plus rhuIL-3 or rhuGM-CSF. The suppressive effects of natural MIP-1 and rMIP-1 alpha were also apparent on a population of BFU-E, CFU-GEMM, and CFU-GM present in cell-sorted fractions of human bone marrow (CD34 HLA-DR+) highly enriched for progenitors with cloning efficiencies of 42% to 75%. These results, along with our previous studies, suggest that MIP-1 alpha, -1 beta, and -2 may have direct myelopoietic enhancing activity for mature progenitors, while MIP-1 alpha may have direct suppressing activity for more immature progenitors.

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.

Ocular herpes simplex virus type 1 (HSV-1) shedding from the latently infected rabbit was induced by iontophoresis of 0.01% epinephrine into the eye. The iontophoresis of epinephrine was at 0.8 mAmp for 8 min once a day for 3 consecutive days. Shedding was determined by the presence of HSV-1 in the tear film obtained with eye swabs. Unilateral epinephrine iontophoresis performed 60 days after inoculation of the virus resulted in ipsilateral HSV-1 shedding in all cases (7/7). Bilateral epinephrine iontophoresis performed on selected days during 170 to 365 days after inoculation resulted in HSV-1 shedding in 75% of the eyes (21/28) and 100% of the rabbits (14/14). All shedding was initiated within 3 days after the third treatment with epinephrine iontophoresis. The shedding frequency induced by epinephrine iontophoresis was significantly higher (pb less than 0.05) than that induced by the other methods employed. HSV-1 was detected in one or both cocultivated explants of trigeminal and superior cervical ganglia for every eye in all experimental groups, indicating that all eyes had the potential to shed. In conclusion, epinephrine iontophoresis induced ocular HSV-1 shedding reliably and with a high frequency in the latently infected rabbits. Furthermore, we suggest that this easily reproducible model of viral shedding offers a system for studying the factors involved in recurrent HSV-1 ocular infections.

Cathodal (-) iontophoresis of 9-beta-D-arabinofuranosyl-adenine 5'-monophosphate (vidarabine monophosphate; Ara-AMP) was performed once daily for 3 days for the treatment of experimental herpes simplex virus type 1 (HSV-1) keratitis in rabbit eyes, and the therapeutic efficacy was compared with that of topical treatment of Ara-AMP and idoxuridine (IDU) administered five times daily for 4 days. With the treatment initiated 24 hr after viral inoculation, Ara-AMP cathodal iontophoresis resulted in significant suppression of epithelial and anterior segment disease processes. Topical IDU (0.5%) or Ara-AMP (10%) also significantly improved the disease process when compared to the placebo-treated group; however, iontophoresis of Ara-AMP resulted in a more marked improvement. Slit-lamp examination indicated that iontophoresis did not cause any observable pathologic changes in corneal epithelium, stroma, conjunctiva, or iris of rabbit eyes. This experiment suggests that iontophoresis of Ara-AMP is a safe and effective approach for preventing the development of herpes simplex keratitis in rabbits.

Iontophoresis of 0.01% levo(-) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days induces ocular shedding of herpes simplex virus type 1 (HSV-1) in latently infected rabbits. In the present experiment, we tested dextro(+) and levo(-) epinephrine for their comparative effects on induced HSV-1 ocular shedding. One hundred percent of the eyes shed virus after either 0.01% dextro(+) or levo(-) epinephrine iontophoresis (8 min, 0.8 mAmp). However, the shedding frequency caused by 0.005% levo(-) epinephrine was significantly higher (P less than 0.05) than that by 0.005% dextro(+) epinephrine when the iontophoresis was conducted at 0.4 mAmp for 4 min. Iontophoresis of 0.001% levo(-) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days and iontophoresis of 0.001% dextro(+) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days did not induce HSV-1 ocular shedding in latently infected rabbits. The data suggest that the mechanism of induction of HSV-1 ocular shedding by epinephrine is correlated to the receptor potency of levo(-) epinephrine.