bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon gamma and lipopolysaccharide promote macrophage adherence to basement membrane glycoproteins.
Authors: Shaw L M , Mercurio A M
Published: June 2004

The ability of thioglycollate (TG)-elicited peritoneal macrophages, a population of recently recruited monocytes, to adhere to the basement membrane glycoproteins laminin and type IV collagen is not a constitutive function of these cells. Adherence can be induced, however, by treatment with IFN-gamma and LPS. In general, IFN-gamma is more potent than LPS in promoting this adherence. Maximal adherence, however, is observed when IFN-gamma (greater than or equal to 5 U/ml) is used together with LPS (2.0 ng/ml). These requirements parallel the conditions needed to obtain tumoricidal activation of TG-elicited macrophages. Adherence to laminin, in the presence of these stimuli, is transient, being maximal at 8 h after their addition and diminishing with longer periods of incubation. In contrast, adherence to type IV collagen does not appear to be transient and IFN-gamma and LPS induce a more prolonged association of macrophages with this substratum.
Selective loss of beta 2-microglobulin mRNA in human colon carcinoma.
Authors: Momburg F , Koch S
Published: June 2004

Expression of MHC class I antigens requires the intracellular assembly of HLA-A,B,C H chains and beta 2-microglobulin (beta 2m). We have investigated the expression of free H chain, beta 2m, and their mRNAs in tissue sections of colon carcinomas that were defective for the native two-chain molecule. In all of these tumors, beta 2m protein and mRNA were found to be completely absent from the neoplastic cells, whereas free H chains and their respective mRNAs were present in abundance in the tumor cell cytoplasm. The selective abrogation of beta 2m expression represents a unique mechanism leading to a complete loss of class I antigen surface expression in vivo.
Clonal expansion of human T lymphocytes initiated by dendritic cells.
Authors: Langhoff E , Steinman R M
Published: June 2004

The accessory cell requirements for cloning T cells in the presence of lectin and T cell growth factors were examined with cells from human peripheral blood. We found that dendritic cells were active and perhaps essential. Single CD4+ lymphocytes could be cloned with 80% efficiency, and CD8+ cells with 50-60% efficiency if 10(3) syngeneic or allogeneic dendritic cells were added. Some T cell clones developed even with one dendritic cell. Monocytes or B lymphocytes from blood were at least 100-fold weaker in supporting clonal growth. These findings suggest a specialized feeder cell requirement, namely dendritic cells, for cloning T lymphocytes from single resting precursors.
Infection of rabbits with human immunodeficiency virus 1. A small animal model for acquired immunodeficiency syndrome.
Authors: Kulaga H , Folks T , Rutledge R , Truckenmiller M E , Gugel E , Kindt T J
Published: June 2004

Injection of rabbits with a human T cell line infected with HIV-1 caused seroconversion within 6 wk, and HIV-1 could be isolated from PBL cultures of infected rabbits. Identity of the isolated virus with HIV-1 was shown by analysis of products amplified by the polymerase chain reaction. HIV-1 infection was seen in rabbits injected with HIV-1-infected cells alone as well as in those that were first infected with HTLV-1 and subsequently with HIV-1. There were no consistent signs of disease in the rabbits infected with HIV-1 alone but HTLV-1/HIV-1-infected rabbits showed signs of illness including diarrhea and weight loss, transient neurologic impairment and, in one animal, a rapidly progressing mammary adenocarcinoma. Examination of organs taken at necropsy from both HIV-1- and HTLV-1/HIV-1-infected animals showed splenic hyperplasia and lymphocyte infiltration of the lungs, as well as moderate damage to liver and kidney in some cases.
The complex pattern of cytokines in serum from patients with meningococcal septic shock. Association between interleukin 6, interleukin 1, and fatal outcome.
Authors: Waage A , Brandtzaeg P , Halstensen A , Kierulf P , Espevik T
Published: June 2004

Serum samples from patients with meningococcal disease were examined for the presence of IL-6, TNF-alpha, and LPS. Median serum concentration of IL-6 was 1,000 times higher in patients with septic shock (189 ng/ml) than in patients with bacteriaemia, meningitis, or combined septic shock and meningitis. 11 of 21 patients with serum levels greater than 3.0 ng/ml died, whereas all 58 patients with serum levels at less than or equal to 3.0 ng/ml, survived. All four patients with serum IL-6 levels greater than 750 ng/ml, died. IL-1 was detected in serum from three patients who also had high serum levels of IL-6, TNF-alpha, and LPS, and rapidly fatal courses. IL-6 appeared to be released into serum later than TNF-alpha, and was detected in serum for up to 36 h. The half-life of IL-6 and TNF-alpha was calculated to be 103 +/- 27 min and 70 +/- 11 min, respectively. These data indicate that a complex pattern of cytokines exists in serum from patients with meningococcal septic shock, and that the release of IL-6 and IL-1, in addition to TNF-alpha, is associated with fatal outcome.
Establishment of two interleukin 6 (B cell stimulatory factor 2/interferon beta 2)-dependent human bone marrow-derived myeloma cell lines.
Authors: Shimizu S , Yoshioka R , Hirose Y , Sugai S , Tachibana J , Konda S
Published: June 2004

Two IL-6-dependent human multiple myeloma cell lines, ILKM2 and ILKM3, were established from the bone marrow of patients with IgG-K multiple myeloma. Both cell lines had the typical morphology and immunocytochemical features of myeloma cells. The surface phenotype of both cell lines was PCA-1+, OKT10+, CD10(J-5)-, CD19(B4)-, CD20(B1)-, CD21(B2)-, and OKIa-1-. A monoclonal cytoplasmic Ig, IgG-K or K L chain, was positive in ILKM2 or ILKM3, respectively. EBV nuclear antigen was negative in both cell lines. They proliferated in the presence of macrophages or macrophage-derived factors (MDF). Among the recombinant cytokines examined, IL-6 most strongly augmented the growth of both cell lines. The anti-IL-6 antibody completely inhibited the IL-6-dependent growth and almost completely inhibited the MDF- or purified MDF-dependent growth of both cell lines, ILKM2 and ILKM3 are now being maintained in the culture medium containing 2 ng/ml rIL-6. These results suggest that IL-6 produced by macrophages may play an important role in the growth of myeloma cells in vivo and that macrophages or IL-6 can be used for establishing human myeloma cell lines.
Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer.
Authors: Sollid L M , Markussen G , Ek J , Gjerde H , Vartdal F , Thorsby E
Published: June 2004

Typing of DNA from 94 unrelated children with celiac disease (CD) with HLA-DQA1 and -DQB1 allele-specific oligonucleotide probes revealed that all but one (i.e., 98.9%) may share a particular combination of a DQA1 and a DQB1 gene. These genes are arranged in cis position on the DR3DQw2 haplotype and in trans position in DR5DQw7/DR7DQw2 heterozygous individuals. Thus, most CD patients may share the same cis- or trans-encoded HLA-DQ alpha/beta heterodimer.
Modulation of HLA class II antigen expression by transfection of sense and antisense DR alpha cDNA.
Authors: Lotteau V , Sands J , Teyton L , Turmel P , Charron D , Strominger J L
Published: June 2004

In the human there are three isotypic forms of MHC class II gene products (HLA-DR, -DQ, and -DP). The isotype-matched alpha-beta dimers are predominant but isotype-mismatched dimers can also be expressed (DR alpha-DQ beta). Here it is shown that the expression of the DR alpha-DQ beta dimer can be correlated to a high ratio of DR alpha/DR beta mRNA. The DR alpha chain expression was modulated by transfection of a sense and antisense DR alpha cDNA. Overexpression of DR alpha promoted the appearance of the DR alpha-DQ beta dimer. On the other hand, pre-existing DR alpha-DQ beta dimer expression was suppressed after antisense DR alpha cDNA transfection. Therefore, imbalanced expression of the alpha and beta chain from a given isotype could lead to the modification of HLA class II phenotype.
Febrile temperatures can synchronize the growth of Plasmodium falciparum in vitro.
Authors: Kwiatkowski D
Published: June 2004

To investigate the possibility that the host fever response in malaria may affect parasite development, we studied the effect of temperature on Plasmodium falciparum in erythrocytic culture in vitro. Growth was markedly suppressed at 40 degrees C compared with 37 degrees C, due to disruption of the second half of the 48-h erythrocytic cycle. However, young intraerythrocytic parasites, which are highly exposed to fever during natural infection, appeared to develop normally at 40 degrees C. Because of the differential temperature sensitivity within the erythrocytic cycle, asynchronous cultures could be synchronized by transient elevations of temperature. Pronounced synchronization was observed when cultures were exposed to periodic elevations of temperature that simulated the 48-h fever cycle of tertian malaria. These findings indicate that malaria fever might act to promote parasite synchronization in vivo.
Diversity and organization of human T cell receptor delta variable gene segments.
Authors: Hata S , Clabby M , Devlin P , Spits H , De Vries J E , Krangel M S
Published: June 2004

Previous studies of the human TCR-delta gene identified a single commonly used V delta segment, denoted V delta 1. To better understand the extent of the human TCR-delta V gene repertoire, TCR-delta transcripts and gene rearrangements were examined in a new panel of cloned human TCR-gamma/delta lymphocytes. Through this analysis we identified and determined the structures of two new V delta segments, denoted V delta 2 and V delta 3. These V delta segments are different from previously characterized V alpha segments, supporting the notion that the human V delta and V alpha repertoires are distinct. Examination of V gamma gene segment usage in these cells reveals that the V delta 2 gene segment is used in conjunction with the V gamma 2 gene segment. Blot hybridization indicates that the V delta 2 gene segment lies between V delta 1 and D delta-J delta-C delta, and within 100 kb of the latter. Analysis of genomic clones indicates that the V delta 3 gene segment lies in an inverted orientation, approximately 2 kb 3' of C delta. This implies that rearrangement of V delta 3 to D delta-J delta-C delta occurs by inversion. Together with previous mapping studies, these results indicate that human V delta segments are dispersed, rather than clustered, within the TCR-alpha/delta locus. The analysis of rearrangements in polyclonal thymocyte DNA suggests that there may be a limited number of additional V delta gene segments yet to be characterized.
Antibacterial activity of human natural killer cells.
Authors: Garcia-Peñarrubia P , Koster F T , Kelley R O , McDowell T D , Bankhurst A D
Published: June 2004

The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass-adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11-enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16-24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing.
Interactions between receptors for interleukin 2 and interleukin 4 on lines of helper T cells (HT-2) and B lymphoma cells (BCL1).
Authors: Fernandez-Botran R , Sanders V M , Vitetta E S
Published: June 2004

IL-2 and IL-4 induce a synergistic proliferative response in HT-2 cells, suggesting that IL-2Rs and IL-4Rs may interact. The purpose of this study was to examine the effect of IL-4 on the expression and function of IL-2Rs. Preincubation of HT-2 and BCL1-3B3 cells with IL-4 for 60 min at 4 degrees C or 37 degrees C resulted in a partial decrease in the number, but not the affinity of high affinity IL-2Rs as evidenced by Scatchard analysis of binding data. The decrease in the number of high affinity receptors correlated with decreased internalization of IL-2. After preincubation with IL-4, crosslinking of 125I-IL-2 to high affinity IL-2Rs also demonstrated a approximately 50% reduction in the number of high affinity IL-2Rs. Another lymphokine, IL-1, which acts on HT-2 cells, had no measurable effect on the affinity or number of IL-2Rs. Taken together, these results indicate that IL-4 downregulates the expression of high affinity IL-2Rs on some cells. It is not known whether this occurs by a direct ligand-mediated receptor interaction, by the sharing of a common receptor subunit, or by interaction of the two receptors with another membrane molecule or cytoskeletal component.
Diversity and structure of human T cell receptor delta chain genes in peripheral blood gamma/delta-bearing T lymphocytes.
Authors: Takihara Y , Reimann J , Michalopoulos E , Ciccone E , Moretta L , Mak T W
Published: June 2004

We have investigated the diversity and repertoire of human TCR delta chain variable gene segments in the human peripheral blood CD4- CD8- (double-negative) population, using rearrangement and expression studies and sequence analyses. 20 TCR delta DNA clones were derived from the RNA of bulk-cultured double-negative T cells and their nucleotide sequences determined. These clones can be classified into six different V delta subfamilies. The distribution, however, was uneven in these cells, with 16 of 20 being derived from the V delta 1 (9) and V delta 2 (7) subfamilies. The remaining subfamilies, V delta 3, V delta 4, V delta 5, and V delta 6, were only represented by one clone each. The majority of these subfamilies seem to consist of a single member, in contrast with the closely linked V alpha subfamilies, which, in most cases, consist of multiple members. Our findings suggest that only a limited number of V delta genes are used in human peripheral blood double-negative T cells and that two major V delta subfamilies (V delta 1 and V delta 2) are used more frequently. Sequence comparison of our cDNA clones to V alpha clones indicates that there is no overlap in usage of V alpha and V delta gene segments, except for the V delta 4 (V alpha 6) subfamily. Comparison of the different V delta sequences suggests that the majority of the sequence diversity is concentrated in the junctions between V, D, and J segments and results from extensive N region diversity.
Spontaneous recovery of rats from experimental allergic encephalomyelitis is dependent on regulation of the immune system by endogenous adrenal corticosteroids.
Authors: MacPhee I A , Antoni F A , Mason D W
Published: June 2004

Lewis rats with experimental allergic encephalomyelitis (EAE), induced either by the subcutaneous injection of guinea pig myelin basic protein (MBP) or by the adoptive transfer of MBP-primed spleen cells, suffer from a single episode of paralysis from which they recover spontaneously. Animals developing EAE were found to have greatly elevated levels of corticosterone in the blood. This endogenous increase in steroid production was accompanied by lymphopenia and depressed delayed-type hypersensitivity responses to OVA, indicating that rats with EAE are immunosuppressed in an antigen-nonspecific fashion. Adrenalectomized rats given subcutaneous implants of corticosterone to maintain basal steroid levels invariably died when EAE was induced. However, if the steroid replacement therapy was adjusted to mimic the hormone levels that were observed in intact rats developing EAE, then the disease followed a nonfatal course closely resembling that seen in the nonadrenalectomized controls. Replacement therapy that achieved serum corticosterone levels slightly higher than those found in intact rats with EAE virtually suppressed the disease completely. It is concluded that endogenous corticosterone release in rats with EAE plays an essential role in the spontaneous recovery that is observed in this condition. However, the subsequent refractory phase that is characteristic of rats that have recovered from EAE induced by active immunization with MBP is not associated with chronically elevated corticosterone levels. This finding is discussed in the light of other data that suggest that unlike the spontaneous recovery, the refractory state has an immunological basis rather than an endocrinological basis.
Lack of expression of HLA-B27 gene in transgenic mouse trophoblast. Conserved genetic pressures underlying extra-embryonic development.
Authors: Oudejans C B , Krimpenfort P , Ploegh H L , Meijer C J
Published: June 2004

The mechanisms that regulate developmental control of the expression of MHC class I genes during generation of extra-embryonic tissues are largely unknown. In the present study, we studied the levels of transcripts of the human HLA-B27 gene in extra-embryonic tissues of transgenic mice containing the HLA-B27 (heavy chain) gene by in situ hybridization with biotinylated single-stranded RNA probes. In contrast to extra-embryonic stromal cells and embryonic tissues which contain (varying levels of) messenger RNA coding for HLA-B27, specific transcripts were not detected in labyrintho-, or spongiotrophoblast, nor in trophoblastic giant cells. These cells are devoid of HLA A and B locus class I transcripts in man. Regulation of expression of human MHC class I genes in extra-embryonic trophoblast in transgenic animals is thus under conserved selective pressure that is retained across a species barrier. Thus, in extra-embryonic tissues, regulation of expression of MHC class I genes is distinct from the mechanisms operating in developing embryonic cells.
Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.
Authors: Meek K , Hasemann C , Pollok B , Alkan S S , Brait M , Slaoui M , Urbain J , Capra J D
Published: June 2004

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.
Fc gamma R(CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2+ in Fc gamma R(CD16)-induced transcription and expression of lymphokine genes.
Authors: Cassatella M A , Anegón I , Cuturi M C , Griskey P , Trinchieri G , Perussia B
Published: June 2004

In this study, we present evidence that interaction of Fc gamma R(CD16) with ligands (immune complexes or anti-CD16 antibodies) induces a rapid rise in [Ca2+]i and fast production of both inositol 1,4,5 triphosphate (IP3) and IP4 in homogeneous NK cell preparations. Part of the initial [Ca2+]i rise observed upon stimulation of NK cells with either anti-CD16 antibodies alone or after their crosslinking at the cell membrane depends on Ca2+ mobilization from intracellular stores, but sustained [Ca2+]i levels are maintained, after the initial spike, through influx of extracellular Ca2+. The [Ca2+]i rise is mediated, at least in part, by increases in IP3 after receptor-induced hydrolysis of membrane polyphosphoinositides (PPI). The role of extracellular Ca2+ in Fc gamma R(CD16)-dependent induction of lymphokine gene expression has been tested by evaluating production, mRNA accumulation and transcription of IFN-gamma and TNF in NK cells stimulated with Fc gamma R(CD16) ligands and/or rIL-2 in the presence of EGTA. Under these conditions, accumulation and transcription of both IFN-gamma and TNF mRNA induced by CD16 ligands, but not that induced by rIL-2, is completely abolished and neither cytokine can be detected at significant levels in the supernatant fluids of cells so treated. These data confirm that NK cell activation by specific ligands occurs through mechanisms distinct from those induced by IL-2, and indicate that extracellular Ca2+ represents a stringent requirement for cytokine production induced in NK cells through specific (Fc gamma R) stimulation. Our data also indicate that the [Ca2+]i rise induced upon Fc gamma R(CD16) crosslinking, though necessary, is not sufficient per se to induce activation of lymphokine genes, compatible with the hypothesis that Fc gamma R(CD16) crosslinking generates additional transducing signals that synergize with IL-2 to maximally activate NK cells.
A unique antigenic epitope of human melanoma is carried on the common melanoma glycoprotein gp95/p97.
Authors: Furukawa K S , Furukawa K , Real F X , Old L J , Lloyd K O
Published: June 2004

Analysis of antibodies present in the serum of melanoma patient FD has shown that they detect a unique tumor epitope present only on the autologous melanoma cell line SK-MEL-131. Previous results had shown that the unique FD epitope is carried on a common glycoprotein of approximately 90 kD, widely expressed on melanoma and a few other cell types. We now show by sequential radioimmunoprecipitation and partial amino acid sequencing that this common molecule is a previously recognized melanoma antigen, originally identified by mouse mAbs, designated gp95 or p97 (and also known as melanotransferrin). Thus, FD is the first of the class I (unique) melanoma antigens that has been characterized and related to a known cell surface molecule.
Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide.
Authors: Stuehr D J , Gross S S , Sakuma I , Levi R , Nathan C F
Published: June 2004

L-arginine-dependent synthesis of nitrite (NO2-) and nitrate (NO3-) by macrophages correlates with and is required for their execution of nonspecific cytotoxicity toward some tumor cells and microbes. However, the bioactive L-arginine metabolites responsible for cytotoxicity are unknown. Mammalian endothelial cells have recently been shown to release nitric oxide (NO.); we therefore determined if this reactive metabolite was synthesized by activated murine macrophages. Macrophage-derived NO. was detected by two independent methods: a bioassay for NO.-mediated relaxation of preconstricted rings of rabbit aorta; and a spectroscopic measurement of the reaction of NO. with clostridial ferredoxin, an Fe-S protein. After activation with IFN-gamma and LPS, macrophages continuously secreted a substance that relaxed rabbit aortic rings denuded of endothelium. Production of the vasorelaxant was enhanced by 0.5 mM L-arginine and inhibited reversibly by NG-methylated L-arginine analogs that block macrophage NO2-/NO3- synthesis. The vasorelaxant was scavenged by ferrous myoglobin, was labile, and was neither NO2- nor a cyclooxygenase metabolite. Activated M phi also secreted a substance that bleached Fd, a reaction carried out by NO. and NO2, but not NO2-. Macrophage bleaching of Fd correlated directly with time, cell number, and concomitant NO2-/NO3- production, required L-arginine, and was independent of reactive oxygen intermediates. Thus, activated murine M phi release NO. and/or a closely related, highly reactive nitrogen oxide such as NO2, during their conversion of L-arginine to NO2-/NO3-. NO. and NO2 may mediate L-arginine-dependent pathologic effects of M phi, as well as physiologic effects not previously considered for this widely distributed cell type.
Regulation of macrophage functions by L-arginine.
Authors: Albina J E , Caldwell M D , Henry W L , Mills C D
Published: June 2004

Sites of inflammation with prominent macrophage infiltration, such as wounds and certain tumors, are uniquely deficient in free arginine. The effects of arginine availability on macrophage physiology were investigated. When cultured in media containing less than 0.1 mM L-arginine, rat resident peritoneal macrophages exhibited enhanced spreading, tumor cytotoxicity, superoxide production, phagocytosis, and protein synthesis. Thus, arginine concentrations similar to those found in sites of inflammation can augment macrophage functions, while those found in plasma (approximately 0.1 mM) and in commonly used culture media (0.4 to 1.2 mM) are inhibitory. Culture in homoarginine, but not D-arginine, ornithine, citrulline, urea, histidine, or lysine also inhibited macrophage tumor cytotoxicity, indicating the specificity of the effect. In contrast to resident macrophages, the tumor cytotoxicity of peritoneal macrophages obtained after C. parvum injection was suppressed by culture in arginine-deficient media. However, L-arginine-deficient media enhanced all other activation-associated functions in C. parvum-elicited macrophages as in resident cells. Arginine-free wound fluid promoted resident macrophage tumoricidal activity when compared with rat serum, and again, the addition of L-arginine was inhibitory. The marked effects of L-arginine availability on macrophage functions, together with the knowledge that these cells modify the extracellular arginine concentration in sites of inflammation through arginase, provide evidence for an autoregulatory mechanism of macrophage activation.
Elastase inhibitor. Characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils.
Authors: Remold-O'Donnell E , Nixon J C , Rose R M
Published: June 2004

A fast-acting inhibitor of serine elastase has been detected at high levels in human neutrophils, fresh monocytes, matured monocytes, and macrophages. The elastase inhibitor was isolated from large scale cultures of the monocyte-like cell line U937 by DNase chromatography, disulfide exchange, Phenyl-Sepharose, Red A-agarose, and DEAE HPLC chromatography with an average yield of 480 micrograms from 1.8 x 10(10) cells. The isolated polypeptide was verified as elastase inhibitor by its ability to (a) form a covalent complex with elastase; and (b) inhibit the elastinolytic activity of elastase. The purified elastase inhibitor molecule is unique, i.e., physiochemical and/or functional properties distinguish it from all other serine proteinase inhibitors. Treatment with iodoacetamide abrogates the ability of the molecule to form a complex with elastase, thereby providing evidence for the presence of an essential cysteine residue. Based on functional criteria, this elastase inhibitor has been grouped with the proteinase inhibitors of the serpin superfamily. The purified elastase inhibitor is a single polypeptide of Mr approximately 42,000. The NH2 terminus appears to be blocked. Compositional analyses indicates five cysteine residues per molecule of approximately 360 amino acid residues. Negligible levels of carbohydrate were detected on gas-liquid chromatography. This finding and the insensitivity of the molecule to peptide N-glycosidase F treatment strongly indicate that the elastase inhibitor is a nonglycosylated protein.
Blast-1 possesses a glycosyl-phosphatidylinositol (GPI) membrane anchor, is related to LFA-3 and OX-45, and maps to chromosome 1q21-23.
Authors: Staunton D E , Fisher R C , LeBeau M M , Lawrence J B , Barton D E , Francke U , Dustin M , Thorley-Lawson D A
Published: June 2004

Blast-1 is a human activation-associated glycoprotein expressed on the surface of leukocytes. Analysis of a translated sequence from a Blast-1 cDNA reveals a single hydrophobic sequence which could traverse the plasma membrane, but is devoid of charged residues that might represent a cytoplasmic tail. Consistent with this characteristic, Blast-1 is demonstrated here to be anchored to the cell surface through a glycosyl-phosphatidylinositol (GPI)-containing lipid. Comparison of Blast-1 to other GPI-anchored membrane proteins revealed a striking primary and secondary structure similarity with MRC OX45 and the lymphocyte function antigen LFA-3. The degree of overall amino acid sequence homology reveals that OX45 is a rat homologue of Blast-1. The greatest homology to LFA-3 occurs between their NH2-terminal Ig-like domains. Evidence is presented that demonstrates that Blast-1 and LFA-3 possess a disulfide-bonded second domain. These common characteristics demonstrate a structural and evolutionary relationship between Blast-1, OX45, LFA-3, and CD2, which in turn suggests a functional role for Blast-1 in cell-cell interactions in the immune response. The gene for Blast-1 has been localized to chromosome 1 q21-q23, indistinguishable from the CD1 cluster of Ig superfamily genes, raising the possibility that they may be linked.
Mature T-lineage leukemia with growth factor-induced multilineage differentiation.
Authors: Griesinger F , Arthur D C , Brunning R , Parkin J L , Ochoa A C , Miller W J , Wilkowski C W , Greenberg J M , Hurvitz C , Kersey J H
Published: June 2004

We report an acute T-lymphoblastic leukemia with a predominantly mature CD3+ CD7+ WT31+ phenotype that was induced to differentiate into different cell lineages by various recombinant human growth factors. In the presence of IL-3 or GM-CSF, the leukemic cells gave rise to myeloid and monocytic cells including terminally differentiated, partially functional, segmented neutrophilic granulocytes as assessed by morphologic, cytochemical, immunophenotypic, and functional criteria. In the presence of IL-2, leukemic granulated lymphoid cells exhibiting MHC-unrestricted cytotoxicity and expressing a CD2+ CD3+ CD5+ CD7+ CD8+ CD33+ WT31+ Leu19+ phenotype arose. Leukemic cell cultures initiated with IL-3 yielded growth factor-independent cells with a mixed lineage phenotype and morphologic and cytochemical evidence of immature blasts. These were T lymphocyte and myeloid surface antigen (CD2,CD3,CD5,CD7,CD13,CD33,WT31) positive. Identical rearrangements of the constant region of the TCR-delta gene and of the joining regions of the TCR-beta, -gamma, and -delta genes were observed in the fresh and all cultured leukemic cells, indicating that they were derived from the same malignant clone. Consistent with the molecular genetic data, the cytogenetic analyses of the GM-CSF-, IL-3-cultured and the growth factor-independent leukemic cells showed the presence of multiple, closely related abnormal clones, all of which had an interstitial deletion of part of the long arm of chromosome 6 and a complex 1;10;12 translocation. In conclusion, these data demonstrate the involvement of a multipotent leukemic precursor cell in this predominantly mature CD2+ CD3+ CD5+ CD7+ WT31+ T-ALL. This multipotent leukemic precursor may be susceptible to various growth factors and respond with ordered differentiation and maturation.
Interleukin 1 production during accessory cell-dependent mitogenesis of T lymphocytes.
Authors: Bhardwaj N , Lau L L , Friedman S M , Crow M K , Steinman R M
Published: June 2004

We have studied the control and significance of IL-1 production in human leukocyte cultures during accessory cell-dependent, T lymphocyte mitogenesis using sensitive bioassays and immunolabeling techniques. In primary antigen-dependent systems like the MLR, IL-1 production was not detected in accessory cells (monocytes, dendritic cells) or T cells, suggesting that it is not an early product in these responses. However, monocytes could be induced to make IL-1 after interacting with sensitized antigen-specific T cells. Both alloreactive T cell clones or freshly prepared lymphoblasts induced IL-1 provided the monocytes carried the HLA-DR antigens to which the T cells were initially sensitized. Even in these circumstances, dendritic cells and B cells failed to make IL-1. The mechanism whereby activated T cells induce IL-1 in monocytes was explored. Supernatants from cocultures of monocytes and T cells or several recombinant cytokines induced little or no IL-1. A more potent antigen independent pathway of IL-1 induction was identified. IL-1 could be induced in third-party HLA-DR nonspecific monocytes in cocultures of alloreactive T cell clones or blasts and HLA-DR-specific dendritic cells. The induction was factor independent since dendritic cells and T blasts placed in a chamber separate from third-party monocytes by a semipermeable membrane did not induce monocyte IL-1. These results suggest that a cell contact mechanism rather than an IL-1-inducing factor leads to IL-1 production. The role of IL-1 in T cell proliferation was tested with a polyclonal anti-IL-1 antibody. The antibody failed to block the proliferation of primary T cells, or alloreactive T cell clones and blasts stimulated with HLA-specific monocytes or dendritic cells, even though IL-1 in the medium was neutralized.
Interferons and bacterial lipopolysaccharide protect macrophages from productive infection by human immunodeficiency virus in vitro.
Authors: Kornbluth R S , Oh P S , Munis J R , Cleveland P H , Richman D D
Published: June 2004

To determine the effects of immunomodulatory agents upon HIV replication in macrophages, cultured monocyte-derived macrophages were treated with various substances and then infected with a macrophage-tropic strain of HIV-1. Pretreatment with rIFN-alpha, IFN-beta, and IFN-gamma, or bacterial LPS prevented viral replication in macrophages. In treated cultures, little or no infectious HIV or p24 core antigen was released into the supernatant, no virions were seen by electron microscopy, no viral RNA or DNA was detectable in the cell lysates, and no cytopathology (as determined by multinucleated giant cell formation) occurred. In contrast, pretreatment with a wide dose range of recombinant IL-1 beta, IL-2, IL-4, IL-6, M-CSF, TNF, or lymphotoxin failed to protect macrophages from productive infection by HIV. A consistent effect of granulocyte/macrophage-CSF on HIV replication in macrophages was not observed. In dose response studies, pretreatment with approximately 100 U/ml of IFN-alpha, approximately 10 U/ml of IFN-beta, or approximately 100 U/ml of IFN-gamma was sufficient to prevent virion release maximally and to prevent cytopathology completely. In kinetic studies, IFN-alpha, IFN-gamma, or LPS were added to the macrophage cultures either before or after infection with HIV. Even when added 3 d after infection with a multiplicity of 1 50% tissue-culture infectious dose per cell, all three treatments markedly reduced virion release, suggesting that these agents act at a point in the viral life cycle beyond the early events of virus binding, penetration, and uncoating. These data indicate that HIV replication in previously uninfected macrophages may be regulated by an inducible host cell mechanism. These findings may explain the restricted replication of HIV in macrophages in vivo and suggest an antiviral role for interferons in the therapy of HIV infection.
Identification of the alternatively spliced exons of murine CD45 (T200) required for reactivity with B220 and other T200-restricted antibodies.
Authors: Johnson P , Greenbaum L , Bottomly K , Trowbridge I S
Published: June 2004

Cell lines expressing specific variants of murine CD45 (T200) were established by infection with retroviral constructs of four cDNAs encoding different forms of CD45. These lines were then used to determine which sequences encoded by alternatively spliced exons of CD45 were required to generate antigenic determinants recognized by anti-CD45 mAbs. The binding of two B220 antibodies (14.8 and RA32C2) to CD45 was dependent on the expression of the first alternatively spliced exon (exon A). A third B220 antibody, RA3-6B2, did not bind to any of the forms of CD45 expressed on fibroblasts. A newly defined anti-CD45R antibody, C363.16A, reacts with an antigenic site dependent upon the expression of the second alternatively spliced exon, exon B.
Identification and characterization of specific receptors for monocyte-derived neutrophil chemotactic factor (MDNCF) on human neutrophils.
Authors: Samanta A K , Oppenheim J J , Matsushima K
Published: June 2004

Specific receptors for a recently purified and cloned monocyte-derived neutrophil chemotactic factor (MDNCF) have been identified on the surface of normal human peripheral blood neutrophils using 125I-labeled recombinant human MDNCF (125I-MDNCF). Competitive binding of 125I-MDNCF to human neutrophils reached a maximal level at 1-3 h at 4 degrees C. The Scatchard analysis showed that there are approximately 20,000 receptors per cell with a single type of high affinity binding (Kd, 8 x 10(-10) M). The receptors for MDNCF are clearly distinct from the receptors for other cytokines and chemotactic agents, e.g., IL-1 alpha, TNF-alpha, and FMLP, C5a, leukotriene B4, and platelet activating factor. Based on the SDS-PAGE analysis of chemically crosslinked 125I-MDNCF receptor complex, there are two polypeptides that bind MDNCF; the molecular weight of these two MDNCF receptors were estimated to be 67,000 and 59,000. Treatment of a promyelocytic cell line, HL60, with 1.25% DMSO for 5 d in vitro increased the number of receptors up to 7,000 receptors/cell with a Kd of 1.2 x 10(-9) M.
Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization.
Authors: Carbone F R , Bevan M J
Published: June 2004

CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.
Translocation t(14;18) in B cell lymphomas as a cause for defective immunoglobulin production.
Authors: de Jong D , Voetdijk B M , Van Ommen G J , Kluin-Nelemans J C , Beverstock G C , Kluin P M
Published: June 2004

Although follicle center cell (FCC) lymphomas represent mature B cells, a considerable percentage do not have detectable Ig production. We have used Southern blotting and the polymerase chain reaction (PCR) to study the involvement of translocations t(14;18) and t(8;14) in causing defective Ig production in 16 Ig- FCC-derived lymphomas and three Ig- B cell acute lymphoblastic leukemias. In 6 of 19 cases, a t(14;18) was present with the other allele either deleted or in germline. In two cases a t(14;18) and a t(8;14) affected both Ig alleles, as confirmed by karyotyping. In two other cases, rearrangement of both bcl-2 on chromosome 18 and c-myc on chromosome 8 were found as well. Although cytogenetic proof was not available, the latter was probably involved in t(8;14). Restriction map analysis of one more case showed rearrangement on the pseudo-JH3 gene on one allele and t(14;18) on the other. Thus, in 11 of 19 cases, defective Ig H chain production could be explained by the inactivation of both Ig H chain genes due to translocation of one allele, in combination with deletions or defective rearrangements of the other allele. In contrast, in 28 of 30 Ig+ lymphomas, one functional Ig H chain allele was found, either in, or not in, combination with t(14;18). In two cases a single rearranged Ig H chain allele was found in combination with rearrangement of bcl-2. No comigration of the single Ig rearrangement with bcl-2, however, was found both by Southern blotting and PCR, suggesting a variant bcl-2 translocation, which leaves the Ig H chain allele functionally intact.
Mixed isotype class II antigen expression. A novel class II molecule is expressed on a murine B cell lymphoma.
Authors: Spencer J S , Kubo R T
Published: June 2004

The structures of Ia molecules expressed by two BALB/c B cell lymphoma lines, A20-1.11 (A20) and 2PK3, were analyzed in an effort to explain the differences in antigen-presenting capacity displayed by these cells. Alloreactive T cell hybridomas specific for I-Ad and antigen-specific, I-Ad-restricted T cells responded well to A20 as the APC. The same alloreactive T cell hybridomas responded weakly or not at all to 2PK3 and the responses of the antigen-specific, I-Ad-restricted T cells were consistently lower to antigen presented by 2PK3 as compared with A20. T cells restricted to I-Ed responded equally well to either A20 or 2PK3 as APC. Additionally 2PK3, but not A20, stimulated a strong syngeneic mixed lymphocyte response. Structural analyses of the Ia antigens revealed that I-A and I-E molecules were expressed by A20, whereas an I-E and a novel I-A-like molecule were expressed by 2PK3. The novel class II molecule was affinity purified from 2PK3 cells using an mAb specific for Ad beta (MK-D6), and this molecule was subsequently shown by an RIA to react with an E alpha-specific mAb (14-4-4S) as well. Chain-specific polyclonal antisera raised against I-A and I-E alpha and beta chains indicated that the 2PK3 "I-A" alpha chain reacted in immunoblot with E alpha-specific and not A alpha-specific antisera, whereas the beta chain reacted with A beta- and not E beta-specific antisera. Peptide map and partial amino acid sequence analyses indicated that the "I-A" molecule expressed by 2PK3 represented a mixed isotype structure resulting from the pairing of Ed alpha with Ad beta. By immunofluorescence staining analysis, 2PK3 did not react with an mAb specific for Ad alpha. 2PK3 was capable of limited antigen presentation through the mixed isotype molecule to I-Ad-restricted OVA-specific T cell hybridomas, although the responses induced were low compared with presentation through I-A on A20. Previous descriptions of the expression of mixed isotype class II molecules in the mouse have resulted primarily from DNA-mediated gene transfer experiments. The results presented indicate that a mixed isotype class II molecule can be expressed naturally.
Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus-specific 57-kD protein.
Authors: Morrison R P , Lyng K , Caldwell H D
Published: June 2004

Recurrent or persistent infections with Chlamydia trachomatis are thought to provide the antigenic stimulus for the chronic inflammation associated with blinding trachoma. We used the guinea pig model of inclusion conjunctivitis to identify chlamydial antigens that may be involved in this deleterious immune response. We purified from chlamydial elementary bodies a genus-specific 57-kD protein that elicited an ocular hypersensitivity response when placed topically onto the conjunctiva of ocular immune guinea pigs. This response was characterized by a predominantly mononuclear macrophage and lymphocyte cellular infiltrate of the submucosal epithelium. The clinical and histological findings were consistent with those of a delayed hypersensitivity response. These data demonstrated that the 57-kD chlamydial protein was a potent stimulator of ocular delayed hypersensitivity. Our findings may be critical to understanding the pathogenesis of the debilitating chlamydial diseases associated with chronic inflammation, such as trachoma and many urogenital syndromes.
Human T cell activation. IV. T cell activation and proliferation via the early activation antigen EA 1.
Authors: Nakamura S , Sung S S , Bjorndahl J M , Fu S M
Published: June 2004

A new mAb G38 was generated against purified EA 1, an early activation antigen. In immunoprecipitation, it was reactive with the same complex precipitated by the initial anti-EA 1 mAb P8. mAb G38 augmented PMA-induced proliferation of PBMC. It was shown to be mitogenic for purified T cells in collaboration with PMA in a dose-dependent manner. This effect was independent of monocytes and other accessory cells. mAb G38 augmented PMA-induced IL-2-R expression. In conjunction with PMA, it induced IL-2 synthesis and secretion. Its effects on IL-2-R and IL-2 expression were documented at both protein and mRNA levels. Both anti-EA 1 mAbs did not induce Ca2+ influx by themselves in PMA-treated T cells. However, the addition of second anti-mouse Ig antibodies induced readily detectable increases in [Ca2+]i. Ca2+-mediated pathways may be utilized as the transduction signal mechanisms. mAb Leu-23 was shown to be reactive with EA 1. mAb Leu-23 was also mitogenic for T cells in the presence of PMA. These findings provide evidence for a functional role for EA 1 in T cell activation and proliferation.