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We present the world-first demonstration of 28-nm embedded MRAM (eMRAM) for frame buffer memory, highlighting the smallest macro size (0.08 mm2/Mb) reported to date. Compared to SRAM that is commonly used for frame buffer memory, eMRAM provides 47% area saving. For frame buffer applications, read disturbance and endurance are the most critical reliability considerations. With magnetic tunnel junction process improvements, we have verified sufficient read disturbance margins and met the endurance requirement (> 1E10 cycles) which corresponds to 10-year continuous usage. Compared to flash-type eMRAM, we have achieved 40% switching current reduction with < 50ns read/write speed.

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.

This study examined the effects of different frequency combinations, 27.12/2 MHz and 60/2 MHz, and the gas flow ratio of dual-frequency capacitively coupled CH 2 F 2 /C 4 F 8 /O 2 /Ar plasmas on the etch characteristics of silicon oxide. The etch rate of the SiO 2 layer decreased and the etch selectivity increased with increasing flow ratio, Q (CH 2 F 2 ), of CH 2 F 2 /(CH 2 F 2 + C 4 F 8 ). The etch rates of SiO 2 and the chemical-vapor deposited amorphous carbon layer decreased with increasing Q (CH 2 F 2 ) but the etch selectivity of SiO 2 over the ACL increased. The etch rate of SiO 2 at 60/2 MHz was faster than that at 27.12/2 MHz. In addition, the line edge roughness and critical dimension values tended to increase with increasing Q (CH 2 F 2 ).

The effects of CH 2 F 2 and N 2 gas flow rates on the etch selectivity of silicon nitride (Si 3 N 4 ) layers to extreme ultra-violet (EUV) resist and the variation of the line edge roughness (LER) of the EUV resist and Si 3 N 4 pattern were investigated during etching of a Si 3 N 4 /EUV resist structure in dual-frequency superimposed CH 2 F 2 /N 2 /Ar capacitive coupled plasmas (DFS-CCP). The flow rates of CH 2 F 2 and N 2 gases played a critical role in determining the process window for ultra-high etch selectivity of Si 3 N 4 /EUV resist due to disproportionate changes in the degree of polymerization on the Si 3 N 4 and EUV resist surfaces. Increasing the CH 2 F 2 flow rate resulted in a smaller steady state CH x F y thickness on the Si 3 N 4 and, in turn, enhanced the Si 3 N 4 etch rate due to enhanced SiF 4 formation, while a CH x F y layer was deposited on the EUV resist surface protecting the resist under certain N 2 flow conditions. The LER values of the etched resist tended to increase at higher CH 2 F 2 flow rates compared to the lower CH 2 F 2 flow rates that resulted from the increased degree of polymerization.

In this study, we compared the line edge roughnesses (LER) and profile angles of chemical vapor deposited (CVD) amorphous carbon ( a -C) patterns etched in an inductively coupled plasma (ICP) etcher produced by varying process parameters such as the N 2 gas flow ratio, Q (N 2 ), and dc self-bias voltage (V dc ) in O 2 /N 2 /Ar and H 2 /N 2 /Ar plasmas. The tendencies of the LER and profile angle values of the etched CVD a -C pattern were similar in both plasmas. The LER was smaller in the O 2 /N 2 /Ar than in the H 2 /N 2 /Ar plasmas, and the profile angle was larger in the O 2 /N 2 /Ar than in the H 2 /N 2 /Ar plasmas under the same processes conditions. The use of O 2 /N 2 /Ar plasma was more advantageous than the H 2 /N 2 /Ar plasma for controlling LER and profile angle.

Highly selective etching of a SiO2 layer using a chemical vapor deposited (CVD) amorphous carbon (a-C) mask pattern was investigated in a dual-frequency superimposed capacitively coupled plasma etcher. The following process parameters of the C4F8/CH2F2/O2/Ar plasmas were varied: the CH2F2/(CH2F2 + O2) flow ratio (Q(CH2F2)), the high frequency power (PHF), and the low frequency power (PLF). It was found a process window exists to obtain infinitely high etch selectivity of the SiO2 layer to the CVD a-C. The process parameters of Q(CH2F2), PHF, and PLF played critical roles in determining the process window for oxide/CVD a-C etch selectivity, presumably due to the disproportionate degree of polymerization on the SiO2 and CVD a-C 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.

In this study, we investigated the etching characteristics of Mo and HfO2 single layers and Mo/HfO2 stacked structure for metal electrode/high-k gate stack patterning in O2/Cl2 inductively coupled plasmas and the effects of O2 addition on the etch rates and etch selectivity of the Mo to the HfO2 layer. By controlling the process parameters such as the O2/Cl2 flow ratio, the top electrode power and the dc self-bias voltage (Vdc), the Mo/HfO2 etch selectivity as high as \cong67 could be obtained. Addition of O2 gas to the O2/Cl2 chemistry improved the Mo/HfO2 etch selectivity because the O2 gas in a certain flow ratio range reduced the HfO2 etching reactions due to less chlorination of Hf but enhanced the Mo etch rate presumably due to effective formation of highly volatile Mo?O?Cl etch by-products.

ZnO and Al-doped ZnO (AZO) were etched in Cl2/CH4/H2/Ar (Cl2-based) and BCl3/CH4/H2/Ar (BCl3-based), inductively coupled plasmas (ICPs) and their etching characteristics were compared by varying the Cl2/(Cl2+CH4) and BCl3/(BCl3+CH4) flow ratios, top electrode power and dc self-bias voltage (Vdc). The etch rates of both ZnO and AZO layers were higher in the Cl2-based chemistry than in the BCl3-based chemistry. The AZO and ZnO etch rates were increased and decreased, respectively, with increasing Cl2 or BCl3 flow ratio. Optical emission measurements of the radical species in the plasma and surface binding states by optical emission spectroscopy (OES) and X-ray photoelectron spectroscopy (XPS), respectively, indicated that, with increasing Cl2 or BCl3 flow ratio; the effective removal of Al in the AZO enhanced the AZO etch rate, whereas the reduced removal of Zn by the Zn(CHx)y products reduced the ZnO etch rate.

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.

Previously, we prepared two different monoclonal antibodies (mAbs) against human 4-1BB (CD137): an agonistic mAb BBK-1 and an antagonistic mAb BBK-2. In this paper, we describe the molecular cloning of these two mAbs and present comparisons of their amino acid sequences. cDNAs encoding the heavy (H) and light (L) chains of the two mAbs were cloned by screening of cDNA libraries constructed from hybridomas secreting these mAbs. Comparisons of amino acid sequences of the two mAbs showed that, while the constant regions of the H and L chains were identical between the two mAbs, the variable region showed 45% identity in H chains and 48% identity in L chains. This suggests that these two mAbs recognize different epitopes of 4-1BB and may have different effects on the activity of 4-1BB.

A new member of human beta-chemokine cDNA was isolated and named leukotactin-1 (Lkn-1). Lkn-1, along with murine macrophage inflammatory protein-related protein-1 and -2, defines a subgroup of beta-chemokines based on two conserved cysteines in addition to the four others conserved in all beta-chemokines. The putative mature Lkn-1 is composed of 92 amino acids with a calculated m.w. of 10,162. The Lkn-1 gene was mapped to human chromosome 17, region q12. Recombinant Lkn-1 was a potent chemoattractant for neutrophils, monocytes, and lymphocytes and induced calcium flux in these cells. Lkn-1 specifically induced calcium flux in CCR1- and CCR3-expressing HOS cell lines. Lkn-1 suppressed colony formation by human granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. Hence, we have isolated and characterized a human C6 beta-chemokine that is a potent agonist at CCR1 and CCR3 and shows broad biologic activities, including leukocyte chemoattraction.

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.

Previously, we and others showed that signals relayed through the murine T cell Ag 4-1BB enhance primary T cell responses, and that blocking the interaction of 4-1BB with its ligand results in decreased responses to polyclonal activators and to alloantigens. Because 4-1BB expression is induced following primary stimulation, we investigated the role of signaling through this molecule in the reactivation of proliferating T cells. To this end, preactivated, 4-1BB-expressing T cells were restimulated in the presence of plate-immobilized mAbs directed against 4-1BB or the prototypic costimulatory molecule CD28. In this work, we show that in the presence of either signal, T cells respond to TCR cross-linking with strong proliferative responses and cytokine production; moreover, our findings indicate that T cell proliferation partially correlates with surface 4-1BB expression. In addition, our results suggest that Ab-mediated costimulatory signals can act independently of potential accessory B7-CD28/CTLA-4 (cytotoxic T lymphocyte Ag-4) interactions. Importantly, the characteristic DNA fragmentation and apoptotic cell death observed after TCR re-engagement are inhibited comparably in the presence of either 4-1BB or CD28 signaling.

This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene. DNase I hypersensitive site (DHS) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence (-7.0 kb) and two DHS in intron 2. The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes. Chloramphenicol acetyltransferase (CAT) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines. A construct termed PFP5a containing -795 bp exhibited the highest CAT activity, and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells. The proximal region in PFP9a20 contained two potential Sp1 binding sites (GC box and GT box) and one Ets binding site (EBS). Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors. When single-point mutation was introduced to each GC box, EBS, and GT box in PFP9a20, at least 3-fold less CAT activity was observed in CTLL-R8 cells. To confirm the importance of the three cis-acting elements in the perforin gene expression, point mutation was introduced again to each proximal GC box, EBS, and GT box of PFP5a. The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity. The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin.

The expression of the murine T cell Ag 4-1BB, a member of the TNF-R family, is induced by T cell activation. Previously, we and others had shown that signaling through 4-1BB enhanced proliferative T cell responses. To investigate a potential role for the interaction of 4-1BB with its ligand (4-1BBL) in T cell activation, we studied the ability of a soluble chimera of 4-1BB (4-1BBFc) to interfere with proliferative responses and cytokine production in models of activation dependent in intercellular interactions. The potential blocking effect of 4-1BBFc was compared with that of the chimeric molecule CTLA-4Ig, a reagent known to interfere with the interaction of CD28 (and/or CTLA-4) with B7 costimulatory receptors. In this study, we report that 4-1BBFc partially blocked both the activation of unfractionated splenocytes triggered by soluble anti-CD3 (anti-CD3s), and the more physiologically relevant responses to alloantigen. In addition, we show that both chimeric molecules partially blocked proliferative responses and IL-2 secretion by highly purified resting T cells activated with anti-CD3s in the presence of fixed accessory cells that express B7 receptors and 4-1BBL. Furthermore, in this model system, the blocking capacity of 4-1BBFc and CTLA-4Ig appears to correlate with the relative expression of their respective cognate receptors (4-1BBL and B7) on the accessory cell. Simultaneous addition of both blocking reagents produced an additive effect in the model systems studied.

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.

4-1BB is an inducible T cell surface receptor which belongs to the nerve growth factor receptor superfamily, a group of cysteine-rich cell-surface proteins. 4-1BB is a 30-kDa glycoprotein and exists as both a monomer and a 55-kDa dimer on the T cell surface. Cross-linking 4-1BB with monoclonal antibody resulted in the 2- to 10-fold enhancement of T cell proliferation. We have isolated and characterized 4-1BB genomic clones and have found that the 4-1BB gene contains two different 5' untranslated regions, which are used alternately to form the 4-1BB mRNA. The two 5' UTRs were encoded in the same chromosome and were separated from one another by an intron of approximately 2.5 kb. The entire gene spans approximately 13 kb of mouse chromosome 4. 4-1BB gene consists of 10 exons and 9 introns, in which there are two exons for 5' untranslated regions and 8 exons for coding region. Most of the putative functional domains were encoded by separate exons. 4-1BB extracellular domain contains four potential C6 (CXn C XX C XX CXn C Xn C) motifs, of which the first motif is partial and the third is distinct from those of nerve growth factor receptor or TNF receptor 1. A comparison of exon-intron organization among the genes of the nerve growth factor receptor family indicated that most C6 motif is interrupted by an intron.

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.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a member of the intercrine/chemokine family which consists of basic, heparin-binding, small molecular weight proteins. We have previously shown that a T cell line, CTLL-R8, carried high-affinity receptors for MIP-1 alpha and the proliferation of CTLL-R8 cells was inhibited by murine recombinant (mr) MIP-1 alpha. We extended our previous studies to murine resting splenic T lymphocytes to determine whether the inhibition of T cell proliferation is a general property of MIP-1 alpha. The resting splenic T cells carried approximately 680 high-affinity binding sites for mrMIP-1 alpha; more than 90% of the primary T cells carried MIP-1 alpha receptors. When the T cells were stimulated with immobilized anti-CD3 mAb in the presence of accessory cells, the MIP-1 alpha binding was reduced. The lowest binding was obtained 2 h after anti-CD3 mAb stimulation due to the internalization of MIP-1 alpha receptors. mrMIP-1 alpha inhibited the anti-CD3 mAb-mediated proliferation of murine splenic T lymphocytes. The maximum inhibition was obtained when mrMIP-1 alpha was added 30 min before anti-CD3 mAb stimulation. Slight inhibition of T cell proliferation was observed when mrMIP-1 alpha was added at the same time as anti-CD3 mAb stimulation. These results indicate that T lymphocytes are regulated negatively by MIP-1 alpha, which occurs when the T cells are exposed to MIP-1 alpha before activation. The negative effect of MIP-1 alpha seems to be mediated in part by the inhibition of IL-2 production, for there was a reduction in both the IL-2 mRNA levels and the IL-2 activity in supernatants from T cells preincubated with MIP-1 alpha before anti-CD3 mAb stimulation.

4-1BB is a 30-kDa inducible T cell Ag, and is expressed predominantly as a 55-kDa dimer on both CD4+ and CD8+ T lymphocytes. The cytoplasmic tail of 4-1BB contains the sequence Cys-Arg-Cys-Pro, which is similar to the sequence Cys-X-Cys-Pro, which mediates the binding of the CD4 and CD8 molecules to the protein tyrosine kinase p56lck. An anti-4-1BB mAb, 53A2, was used to determine whether 4-1BB may associate with p56lck. The 53A2 mAb specifically recognized 4-1BB on a CD8+ T cell line, CTLL-2, and coimmunoprecipitated a 56-kDa protein along with 4-1BB. Peptide mapping indicated that the 56-kDa phosphoprotein was identical to p56lck. The coimmunoprecipitation of p56lck with 4-1BB also occurred in nonlymphoid cells such as insect (Sf-21) and HeLa cells when the two recombinant proteins were coexpressed. Analysis of mutant p56lck recombinant proteins showed that two cysteine residues critical for p56lck-CD4 (or -CD8) complex formation are also required for the p56lck-4-1BB interaction. These studies establish that 4-1BB physically associates with p56lck.

4-1BB is an inducible receptor-like protein expressed in both cytolytic and Th cells. Optimal induction of 4-1BB mRNA in T cells required both PMA and ionomycin stimulation, indicating that protein kinase C activation and increases in intracellular Ca2+ were required for its expression. 4-1BB was categorized as an early activation gene since the protein synthesis inhibitor, cycloheximide, blocked the induction of 4-1BB mRNA. A rat mAb, 53A2, was generated against recombinant soluble 4-1BB and was used to characterize this molecule. 4-1BB is a 30-kDa glycoprotein and appears to exist as both a monomer and a 55-kDa dimer on the cell surface of a T cell clone. The 4-1BB protein may be post-translationally modified since its predicted backbone is 25 kDa. FACS analysis indicated that 4-1BB was inducible and expressed on the cell surface of activated splenic T cells and thymocytes. Cross-linking of 4-1BB on anti-CD3-stimulated T cells with 53A2 resulted in a dramatic enhancement of T cell proliferation. This suggests that 4-1BB may function as an accessory signaling molecule during T cell activation.

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.

Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-1 alpha), a cytokine with myelopoietic activity in vitro, was assessed in vivo by injection into C3H/HeJ mice for effects on proliferation (percentage of cells in S phase DNA synthesis of the cell cycle) and absolute numbers of granulocyte-macrophage, erythroid, and multipotential progenitor cells in the femur and spleen, and on nucleated cellularity in the bone marrow, spleen, and blood. rmuMIP-1 alpha rapidly decreased cycling rates (at 2 to 10 micrograms/mouse i.v.) and absolute numbers (at 5 to 10 micrograms/mouse i.v.) of myeloid progenitor cells in the marrow and spleen. These effects were dose- and time-dependent and reversible. Suppressive effects were noted within 3 to 24 h for cell cycling and absolute numbers of progenitor cells in the marrow and spleen, and by 48 h for circulating neutrophils. A study comparing the effects of i.v. injection of rmuMIP-1 alpha versus rmuMIP-1 beta, a biochemically similar molecule but with no myelosuppressive effects in vitro, demonstrated myelosuppression in vivo by rmuMIP-1 alpha, but not by rmuMIP-1 beta. The results suggest that rmuMIP-1 alpha has myelosuppressive activity in vivo and offers the possibility that it may be a useful adjunct to treatments involving cytotoxic drugs because of its reversible suppressive effects on normal progenitor cell cycling.

We have produced recombinant proteins for a cytokine, L2G25BP (macrophage inflammatory protein-1 alpha) (MIP-1 alpha). By using the recombinant protein (rMIP-1 alpha), receptors for MIP-1 alpha were identified on Con A-stimulated and unstimulated CTLL-R8, a T cell line, and LPS-stimulated RAW 264.7, a macrophage cell line. The 125I-rMIP-1 alpha binds to the receptor in a specific and saturable manner. Scatchard analysis indicated a single class of high affinity receptor, with a Kd of approximately 1.5 x 10(-9) M and approximately 1200 binding sites/Con A-stimulated CTLL-R8 cell and a Kd of 0.9 x 10(-9) M and approximately 380 binding sites/RAW 264.7 cell. 125I-rMIP-1 alpha binding was inhibited by unlabeled rMIP-1 alpha in a dose-dependent manner, but not by IL-1 alpha or IL-2. rMIP-1 alpha inhibited the proliferation of unstimulated CTLL-R8 cells. Rabbit anti-rMIP-1 alpha antibodies blocked the growth-inhibitory effect of the rMIP-1 alpha on CTLL-R8 cells.

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.

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.

In this paper, a compact co-planar waveguide band-pass filter is proposed for ultra-wideband (UWB) communication systems by utilizing perforation in the ground layer. An aperture compensation technique is developed to control the coupling strength, since UWB filters require a strong coupling strength due to its wide bandwidth (> 50%). The coupling strength appears frequency-distributed and its maximum value rapidly rises as the aperture widens. Then, a pair of capacitively loaded line sections is inserted between two identical parallel coupled lines to formulate a new planar band-pass filter with multi-pole band-pass behavior. The predicted and measured results show attractive properties of the proposed filter with a pass-band of 3.1 to 5.0 GHz (50%), |S/sub 11/| < -18 dB, group delay 800 psec, insertion loss < 1 dB, and the size of 9 /spl times/ 9 mm/sup 2/.

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.

Previously, we prepared two different monoclonal antibodies (mAbs) against human 4-1BB (CD137): an agonistic mAb BBK-1 and an antagonistic mAb BBK-2. In this paper, we describe the molecular cloning of these two mAbs and present comparisons of their amino acid sequences. cDNAs encoding the heavy (H) and light (L) chains of the two mAbs were cloned by screening of cDNA libraries constructed from hybridomas secreting these mAbs. Comparisons of amino acid sequences of the two mAbs showed that, while the constant regions of the H and L chains were identical between the two mAbs, the variable region showed 45% identity in H chains and 48% identity in L chains. This suggests that these two mAbs recognize different epitopes of 4-1BB and may have different effects on the activity of 4-1BB.

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.

An understanding of the cellular genes whose expression is altered during HSV reactivation will enable us to better understand host responses and biochemical pathways involved in the process. Furthermore, this knowledge could allow us to develop gene-targeted inhibitors to prevent viral reactivation. Mice latent with HSV-1 strain McKrae and uninfected control mice were subjected to hyperthermic stress (43 degrees C for 10 min) and their trigeminal ganglia (TG) collected 1 h later. Two additional groups included HSV-1 latently infected and uninfected mice not subjected to hyperthermic stress. Poly A+ mRNA was enriched from total mouse TG RNA and reverse transcribed using MMLV RT. Radioactively labeled cDNAs were analyzed by microarray analysis. A stress/toxicology array of 149 mouse genes on a nylon membrane was used. The labeled cDNAs prepared from latently infected, stressed mice demonstrated 3-fold or greater increases in certain mRNA-early response genes (ERGs) compared to cDNAs from uninfected, stressed control mice. The ERG mRNAs that showed increases included two heat shock proteins (HSP60 and HSP40), a basic transcription factor (BTF T62), a DNA repair enzyme, two kinases [MAP kinase and a stress-induced protein kinase (SADK)], an oxidative stress-induced protein, a manganese superoxide dismutase precursor-2 (SOD-2), and cyclooxygenase 2 (COX-2). The gene expression in unstressed, infected TGs was similar to the gene expression in unstressed, uninfected controls. These results suggest that there is a significant difference in the ERG expression profile in latently infected TGs undergoing stress-induced reactivation compared to uninfected TGs.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.

Host immune response is known to contribute to the progression of periodontitis, and alveolar bone destruction in periodontitis is associated with enhanced osteoclast activity. Therefore, we evaluated the roles of activated lymphocyte subsets in osteoclastogenesis. Osteoclast precursors were co-cultured with activated lymphocytes (B, CD4(+) T, CD8(+) T) in the presence of either macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus soluble receptor activator of NF-kappaB ligand (sRANKL), and subsequent differentiation into active osteoclasts was evaluated by a resorption assay. The activated B and CD4(+) cells, but not CD8(+) T cells, induced osteoclast differentiation in the presence of M-CSF alone. In the presence of M-CSF and sRANKL, B cells induced the formation of small but highly active osteoclasts and increased resorption, while CD8(+) T cells profoundly suppressed osteoclastogenesis. Co-culture using an insert well or supernatant suggested that both B and CD8(+) T cells acted on osteoclasts mostly via soluble proteins. Activated B cells expressed many osteoclastogenic factors including RANKL, TNF-alpha, IL-6, MIP-1alpha, and MCP-3. CD8(+) T cells expressed a substantial amount of osteoprotegerin (OPG) along with RANKL. However, blocking antibody to OPG did not reverse the suppression by CD8(+) T cells, suggesting that other factor(s) are involved. Taken together, activated B cells promoted osteoclastogenesis, while CD8(+) T cells inhibited the osteoclast formation via direct interaction. The results imply the importance of lymphocyte subpopulations in the development of periodontitis.

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.

4-1BB is a member of the TNF receptor family predominantly expressed on activated T cells, and binds an inducible ligand found on B cells, macrophages and dendritic cells. Whereas ligation of 4-1BB has been shown to enhance response of purified CD8 T cells to mitogens, and to augment NK activity and generation of cytotoxic T lymphocytes in vivo, there are little direct data on 4-1BB action during CD4 responses. Using pigeon cytochrome c-presenting fibroblast antigen-presenting cells transfected with 4-1BB ligand (4-1BBL), we show that engaging 4-1BB on naive CD4 cells promotes proliferation, cell cycle progression and IL-2 secretion, and suppresses cell death, all to a similar extent as B7-1 engagement of CD28. In addition, 4-1BBL synergizes with B7 and ICAM to enhance naive CD4 proliferation when antigen is limiting. 4-1BBL alone, and to a greater extent with B7, also augmented IL-2 secretion resting antigen-experienced CD4 cells, as typified by T helper clones, whereas short-term effector cells showed similar levels of proliferation and cytokine secretion regardless of whether 4-1BB was engaged. A major role in augmenting IFN-gamma, IL-4 or IL-5 was not demonstrated. Blocking studies with activated B cells presenting antigen showed that 4-1BB participates in promoting IL-2 production by resting CD4 cells, confirming that 4-1BBL can play a role in antigen-specific CD4 T cell responses.

In this report we show that allo-stimulated naïve CD4+ cells when cultured in IL-2, IFN-gamma, IL-12 and anti-IL-4 differentiated into Th1 cells expressing very low amounts of 4-1BB molecule, but high amounts of CD28. By contrast, allo-stimulated naive CD4+ cells cultured in the presence of exogenous IL-2, IL-10, IL-4 and anti-INF-gamma evolved into Th2 expressing high amounts of both 4-1BB and CD28. Various Th1/Th2 clones derived from limiting dilution also exhibited similar expression pattern. This differential expression of co-stimulatory molecules on Th subsets account for the ability of 4-1BB to trigger both the proliferation and production of IL-4 by Th2 cells only and for the ability of CD28 to trigger proliferation and typical secretion in both Th1 and Th2 cells.

The human CC chemokine leukotactin-1 (Lkn-1) is both a strong chemoattractant for neutrophils, monocytes, and lymphocytes and a potent agonist for CCR1 and CCR3. However, human neutrophils do not migrate when the cells are stimulated with other human CC chemokines, such as human macrophage inflammatory protein-1 alpha (hMIP-1 alpha) and eotaxin, which also use the CCR1 and CCR3 as their receptors. In this report, we demonstrate that while hMIP-1 alpha induced a negligible level of calcium flux and chemotaxis, Lkn-1 produced a high level of calcium flux and chemotaxis in human neutrophils. Lkn-1 cross-desensitized hMIP-1 alpha-induced calcium flux, but hMIP-1 alpha had little effect on the Lkn-1-induced response in human neutrophils. The same pattern was observed in peritoneal neutrophils from wild-type mice, whereas neutrophils from CCR1-/- mice failed to respond to either MIP-1 alpha or Lkn-1. Scatchard analysis revealed a single class of receptor for both hMIP-1 alpha and Lkn-1 on human neutrophils with dissociation constants (Kd) of 3.2 nM and 1.1 nM, respectively. We conclude that CCR1 is a receptor mediating responses to both MIP-1 alpha and Lkn-1 on neutrophils and produces different biological responses depending on the ligand bound.