bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene conversion between murine class II major histocompatibility complex loci. Functional and molecular evidence from the bm 12 mutant.
Authors: Mengle-Gaw L , Conner S , McDevitt H O , Fathman C G
Published: June 2004

The experiments presented in this study define the molecular basis of the bm 12 mutation. Initial characterization of an alloreactive T cell clone, 4.1.4, showed this clone to recognize an allodeterminant present on the E beta b and A beta bm12 chains, but not on the bm 12 parent A beta b chain. To define the extent of sequence shared between the I-E beta product and the mutant I-A beta product, we isolated a cDNA clone of the E beta b gene and determined its nucleotide sequence. Comparison of the nucleotide sequences of E beta b, A beta b, and A beta bm12 shows the the A beta bm12 gene to be identical to the E beta b gene in the region where it differs from its A beta b parent. We predict that the bm 12 mutation arose by gene conversion of this region, which spans 14 nucleotides between amino acid residues 67-71 of the mature A beta chain, from the E beta b locus to the corresponding position at the A beta b locus. Recognition of this region, which spans one of the previously defined E beta allelic "hypervariable" regions, by an alloreactive T cell clone provides the first direct evidence of the functional importance of these hypervariable regions in T cell stimulation. The identification of a gene conversion event involving one of these allelic variable regions implicates conversion as a mechanism that acts on class II beta genes to create sequence diversity in regions of Ia molecules that interact with foreign antigen or a T cell receptor, regions where protein sequence polymorphism would presumably be selected for by the expanded ability it affords the organism to mount effective immune responses against a wider variety of foreign antigens.
Increased cytotoxicity of normal rabbit serum for lectin-resistant mutants of animal cells.
Authors: Jones C
Published: June 2004

Plant lectins are cytotoxic and can be used to select for mutants of animal cells that exhibit structural changes in cell surface carbohydrates reflecting glycosylation defects. We isolated eight lectin mutants of Chinese hamster ovary (CHO) cells that appear to represent three different phenotype classes. These lectin mutants were much more sensitive to the cytotoxic action of normal rabbit serum (NRS) than were the parental cells. This increased cytotoxicity was heat sensitive, specifically absorbed, and inhibited by simple and complex carbohydrates. No killing was observed under conditions in which only the alternate complement pathway was active. An NRS-resistant subclone that was isolated from one lectin mutant was shown to have also regained wild type behavior when tested with the lectins. The possibility that naturally occurring antibodies in rabbit serum are reacting with incomplete carbohydrate chains on the surface of the lectin mutants is discussed.
Frequency of expressed immunoglobulin light chain genes in lipopolysaccharide-stimulated BALB/c spleen cells.
Authors: Zhu D , Lefkovits I , Köhler G
Published: June 2004

Variants of four hybridoma lines secreting antibodies specific for either trinitrophenyl (TNP) or sheep erythrocytes (SRBC) were isolated which have lost either the specific heavy (H) chain or the specific light (L) chain. They were fused to lipopolysaccharide-stimulated mouse spleen cells and the resulting secondary hybridomas were screened for the restoration of the original antibody specificity. Antibody activity was 37 times more frequently restored with fusion lines donating H chain than with those donating L chain. We obtained a variety of different (spleen cell derived) L chains in association with one H chain and one specificity. We found that those L chains originally associated with a given H chain were rescued most often. Their frequencies were 1:74 and 1:490 for an anti-SRBC- and an anti-TNP-restoring kappa L chain, respectively. Two most commonly observed V-J kappa combinations in one anti-SRBC complementation group were detected with a 5- and 30-fold reduced frequency in two anti-TNP groups, indicating somatic diversification of kappa chains. It is shown that the Sp7/HK line resumes anti-TNP activity with a mutated, non-germline lambda 1 chain, which was found in 30% of lambda 1-expressing hybridomas.
Dual origin of mouse spleen macrophages.
Authors: van Furth R , Diesselhoff-den Dulk M M
Published: June 2004

The present study concerns the isolation, characterization, origin, and kinetics of spleen macrophages. The spleen was first perfused in situ to remove monocytes from the vascular bed and then dissected and treated with collagenase. The macrophages in the cell suspension thus obtained were characterized morphologically and cytochemically and then quantitated. The spleen cell suspension was incubated for 24 h in Leighton tubes to obtain an enriched glass-adherent population of macrophages for characterization and [3H]thymidine-labeling studies. Almost all of the adhering macrophages were esterase positive, had Fc and C3b receptors, and ingested EIgG and opsonized bacteria. In vitro labeling with [3H]thymidine showed that approximately 5% of the mononuclear phagocytes in the spleen synthesize DNA and must be considered to be dividing cells. The course of the number of labeled monocytes and macrophages after a single injection of [3H]thymidine indicates migration of monocytes into the spleen, where they become macrophages. Calculation of the influx of monocytes into the spleen and of the local production of macrophages by DNA-synthesizing mononuclear phagocytes showed that under steady-state conditions, 55% of the population of spleen macrophages is supplied by monocyte influx and 45% by local production. This means that there is a dual origin of spleen macrophages. The mean turnover time calculated with the value for the efflux of spleen macrophages is 6.0 d.
A multivalent lacto-N-fucopentaose III-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective.
Authors: Fenderson B A , Zehavi U , Hakomori S
Published: June 2004

A multivalent lacto-N-fucopentaose (LNFP) III-lysyllysine conjugate was observed to decompact preimplantation mouse embryos. Decompaction was not obtained with free oligosaccharides (LNFP II and III), nor with multivalent LNFP II-lysyllysine or chitotriose-lysyllysine conjugates. These results suggest a role for X hapten recognition during compaction and suggest further that X hapten valency may play a key role in modulating this developmental process.
Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide-producing enzyme.
Authors: Guthrie L A , McPhail L C , Henson P M , Johnston R B
Published: June 2004

We investigated the capacity of bacterial endotoxin (lipopolysaccharide, LPS) to modify the oxidative metabolic response to membrane stimulation of human neutrophils. Neutrophils were pretreated for 60 min with LPS, 10 ng/ml, then stimulated by exposure to fixed immune complexes, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), or phorbol myristate acetate. Release of superoxide anion (O-2) was up to 7-times greater in cells preincubated with LPS, depending upon the stimulus used. Consumption of oxygen and release of hydrogen peroxide (H2O2) were similarly increased, using FMLP as stimulus. The enhancement was accompanied by a reduction in lag time and an increase in the rate of the response, but the duration of the oxidative events was not changed. The molecular basis for the augmented oxidative response of LPS-pretreated cells was investigated. Preincubation with LPS at 0 degrees C prevented priming, but preincubation in the presence of cycloheximide or chelation of extracellular calcium ion did not. Neutrophils preincubated with LPS had slightly decreased numbers of binding sites and equivalent binding affinity for radiolabeled FMLP. Possible changes in the enzyme responsible for the oxidative burst were analyzed by studying NADPH-dependent generation of O-2 by particulate fractions from cells preincubated with LPS or buffer, then stimulated before cell disruption. The fraction prepared from LPS-pretreated neutrophils exhibited greater release of O-2 over a wide range of concentrations of NADPH. The calculated apparent Km for NADPH was equivalent in the two fractions, but the Vmax was increased 2.5-fold in the subcellular fraction from LPS-pretreated cells. These results suggest that LPS could increase neutrophil-mediated host defense or the tissue damage associated with endotoxemia by enhancing the generation of oxygen metabolites by neutrophils. These results also support the concept that the neutrophil is not an end-stage cell in regard to function or metabolic activity.
Resting and sensitized T lymphocytes exhibit distinct stimulatory (antigen-presenting cell) requirements for growth and lymphokine release.
Authors: Inaba K , Steinman R M
Published: June 2004

Previous studies have shown that unprimed or resting T lymphocytes will grow and release lymphokines when stimulated by dendritic cells (DC). We now have examined the stimulatory requirements for antigen-primed or blast-transformed T cells. The latter were derived from dendritic/T cell clusters that developed during the primary mixed leukocyte reaction (MLR). The specificity of the blasts was established by a binding assay in which most T cells aggregated small B lymphocytes of the appropriate haplotype within 2 h at 4 or 37 degrees C. Since unprimed T cells did not aggregate allogeneic B cells, we suggest that DC induce T lymphocytes to express additional functioning receptors for antigen. Lyt-2-T blasts did not grow or release interleukin 2 or B cell helper factors unless rechallenged with specific alloantigen, whereupon growth (generation time of 14-18 h) and lymphokine release rapidly resumed. The blasts could be stimulated by allogeneic macrophages, B cells, and B lymphoblasts, whereas the primary MLR was initiated primarily by DC. responsiveness appeared restricted to the I region of the major histocompatibility complex, and varied directly with the level of Ia antigens on the stimulator cells. The interaction of B cells and T blasts was bidirectional. The T blasts would grow and form B cell helper factors, while the B cells grew and secreted antibody. However, the efficacy of T cell-mediated antibody formation was enhanced some 10-fold by the addition of specific antigen. Therefore, responses of resting helper T cells, then, are initiated by antigen plus DC. Once sensitized, T blasts interact independently with antigen presented by other leukocytes.
Thymus dictates major histocompatibility complex (MHC) specificity and immune response gene phenotype of class II MHC-restricted T cells but not of class I MHC-restricted T cells.
Authors: Kast W M , de Waal L P , Melief C J
Published: June 2004

Athymic H-2b nude mice received grafts from C57BL/6 (Sendai virus and H-Y antigen cytotoxic T lymphocyte [CTL] responder type), bm1 (H-2Kb mutant, Sendai CTL nonresponder type), or bm12 (H-21-A mutant, H-Y CTL nonresponder type) neonates. In observations of the CTL response to H-Y, both recipients and thymus donors were female. All types of thymus engraftment resulted in mature H-2b splenic T lymphocyte surface phenotype in nude hosts. T cell immunocompetence (as measured by major histocompatibility complex [MHC] CTL responses to allogeneic cells) was restored, and induced nonresponsiveness to the MHC determinants of the engrafted thymus in the nude host. The CTL reaction to Sendai virus in both responder type C57BL/6 and nonresponder type bm1 neonatal thymuses allowed maturation of Sendai-specific, H-2Kb-restricted CTL. For the CTL reaction to H-Y, only responder type C57BL/6 thymuses restored the CTL response, whereas this was not achieved with thymuses from nonresponder type bm12 neonatal females. Results of double thymus (B6 and bm12) engraftment excluded the possibility that this latter effect was caused by suppression. In addition, athymic bm1 mice were engrafted with thymuses from either B6 (Sendai CTL responder type) or syngeneic bm1 neonates (Sendai CTL nonresponder type). Again, both types of neonate thymuses restored T cell competence as measured by MHC/CTL responses to allogeneic cells. However, neither responder B6 nor nonresponder bm1 neonate thymus grafts allowed maturation of Sendai-specific CTL. In conclusion, the thymus dictates MHC specificity and immune response gene phenotype of T cells restricted to class II MHC molecules but not of T cells restricted to class I MHC molecules.
Selective inhibition of growth factor-dependent human B cell proliferation by monoclonal antibody AB1 to an antigen expressed by activated B cells.
Authors: Jung L K , Fu S M
Published: June 2004

A monoclonal antibody, AB1, was established with activated human B cells as immunogen. AB1 stained activated B cells but not activated T cells. Its selective reactivity to activated B cells was further documented by its nonreactivity to activated T cells, resting T and B cells, monocytes, granulocytes, bone marrow cells, leukemic cells, and cells from cell lines of T, B, and myeloid lineages. Upon activation, the antigen appeared on B cells as early as 3-4 h after stimulation and was fully expressed by 38 h. The expression of this antigen was not dependent on the presence of B cell stimulatory factor(s). Anti-IgM antibodies by themselves induced its expression. AB1 inhibited B cell proliferation that was induced by a low dose anti-IgM antibody and conditioned medium containing B cell stimulatory factor. It did not inhibit B cell proliferation induced by either high doses of anti-IgM antibodies or by formalinized Staphylococcus aureus. It also failed to inhibit T cell mitogenesis. The possibility exists that this antigen is related to the receptor for B cell stimulatory factor.
Factor B, the complement alternative pathway serine proteinase, is a major constitutive protein synthesized and secreted by resident and elicited mouse macrophages.
Authors: Sundsmo J S , Chin J R , Papin R A , Fair D S , Werb Z
Published: June 2004

Factor B, the complement alternative pathway serine proteinase, a class III gene product of the major histocompatibility complex, is a major constitutive secretion product of mouse mononuclear phagocytes. This glycoprotein was synthesized and secreted by macrophages as a doublet of Mr 90,000 and 93,000 polypeptides that were immunoprecipitable with antibodies raised to human serum factor B, and that were indistinguishable from plasma factor B by immunoreactivity, peptide mapping, and molecular weight. Macrophage factor B was cleaved and activated to factor Bb- and Ba-like fragments by factor D and cobra venom factor. Some conversion of macrophage factor B to Bb-sized fragments occurred spontaneously in the conditioned culture medium after several hours. Factor B represented approximately 0.5% of newly synthesized protein and 4-6% of the secreted protein of resident peritoneal macrophages and macrophages elicited with thioglycollate broth, pyran copolymer, NaIO4, bacillus Calmette-Guerin, or Corynebacterium parvum. We detected synthesis of factor B immediately upon explanting these macrophages in culture; synthesis continued for several days in culture. The rate of secretion of factor B, as a proportion of total protein secretion in culture, remained constant with time. By radioimmunoassay, factor B antigens accumulated in the 24-h macrophage-conditioned culture medium at 2-10 nM, and was present in cell lysates at 4-8 nmol per 10(6) cells. We detected synthesis of factor B in bone marrow-derived macrophages as early as 5 d of culture. The P388D1 macrophage line synthesized factor B, but mouse L cells did not. In contrast, apolipoprotein E, another secreted protein of macrophages, was secreted by resident and thioglycollate-elicited macrophages but not by freshly harvested pyran copolymer-activated macrophages. Its synthesis was initiated at day 9 in culture of bone marrow-derived macrophages. These data support the classification of factor B as a constitutive biosynthetic and secreted protein of immature and mature macrophages in various states of activation. Production of factor B was modulated by treatment of macrophages in vivo or in culture with bacterial lipopolysaccharide endotoxin, which increased the synthesis, secretion, and accumulation of factor B up to 11-fold.
Antihemostatic, antiinflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini.
Authors: Ribeiro J M , Makoul G T , Levine J , Robinson D R , Spielman A
Published: June 2004

Pilocarpine-induced saliva of the tick, Ixodes dammini, inhibited platelet aggregation triggered by ADP and collagen, as well as platelet-aggregation factor. In addition, we found apyrase activity (which degrades ATP and ADP to AMP and orthophosphate) and an anticoagulant. We showed the presence of prostaglandin E2 (PGE2) by bioassay and radioimmunoassay. This saliva inhibited interleukin 2 production by T cell hybridomas, an activity consistent with that of PGE2. A kininase was demonstrated, and this may counteract the algesia- and edema-promoting properties of PGE2. Together, these salivary components produce antihemostatic, antiinflammatory, and immunosuppressive effects that may facilitate feeding, as well as transmission of tick-borne pathogens.
Pyrimidine salvage in Giardia lamblia.
Authors: Aldritt S M , Tien P , Wang C C
Published: June 2004

We have found that the anaerobic protozoan parasite Giardia lamblia is incapable of de novo pyrimidine metabolism, as shown by its inability to incorporate orotate, bicarbonate, and aspartate into the pyrimidine nucleotide pool. Results from high performance liquid chromatography of pyrimidine and pyrimidine nucleoside pulse-labeled nucleotide pools and enzyme assays suggest that the parasite satisfies its pyrimidine nucleotide needs predominantly through salvage of uracil by a cytoplasmic uracil phosphoribosyltransferase. Exogenous uridine and cytidine are primarily converted to uracil by the action of uridine hydrolase and cytidine deaminase before incorporation into nucleotide pools. Direct salvage of cytosine occurs to a relatively limited extent via cytosine phosphoribosyltransferase. G. lamblia relies on salvage of exogenous thymidine for ribosylthymine monophosphate (TMP) synthesis, accomplished primarily through the action of a 100,000 g-pelletable thymidine phosphotransferase.
Interaction of the heterozygous nude gene with the asplenia trait in mammary tumorigenesis.
Authors: Lopez D M , Pauley R J , Lozzio B B
Published: June 2004

The BALB/c mouse strain has been shown to contain endogenous mouse mammary tumor virus (MMTV) proviral sequences. However, no exogenous MMTV particles have been detected in their tissues. Female BALB/c mice from our colonies exhibit a very low incidence of spontaneous mammary tumors (SMT); less than 1% at up to 20 mo of age. Immunodeficient BALB/c mice heterozygous for the nude gene (nu/+, +/+), for the dominant hemimelia gene associated with asplenia (+/+, Dh/+), or for both traits (nu/+, Dh/+) have been examined for SMT incidence and the presence of MMTV proviruses. Based on restriction digestion with Eco RI, Bam HI, and Pst I, the immunodeficient mice have an MMTV provirus copy number and organization identical to the BALB/cCrgl strain. This MMTV DNA pattern is distinct from the MMTV proviruses in C3H/He, C57BL/6J and CBA/CaJ mice, which were parental strains of the immunodeficient mutants. Normal female BALB/c or BALB/c heterozygous for the asplenic trait do not develop significant numbers of SMT at up to 19 mo of age. In contrast, an incidence of 23.8% and 57.7% SMT was observed in BALB/c nu/+ heterozygotes, and in BALB/c nu/+, Dh/+ heterozygotes, respectively. These results indicate that agenesis of the spleen, concomitant with the presence of the heterozygous nude gene, contribute to a high incidence of SMT in the low-SMT BALB/c mouse strain.
TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors.
Authors: Burns G F , Triglia T , Werkmeister J A , Begley C G , Boyd A W
Published: June 2004

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.
gp72, the 72 kilodalton glycoprotein, is the membrane acceptor site for C3 on Trypanosoma cruzi epimastigotes.
Authors: Joiner K , Hieny S , Kirchhoff L V , Sher A
Published: June 2004

We examined the interaction of complement component C3 with surface molecules on Trypanosoma cruzi. Five- to six-fold more C3 was bound to epimastigotes (Epi) than to metacyclic trypomastigotes (CMT) of strain M88. Epi and CMT were surface iodinated, then incubated in C8-deficient serum, and detergent lysates were applied to anti-C3 antibody that had been coupled to Sepharose. We found that 9.20-10.24% of applied 125I-Epi protein bound to anti-C3-sepharose, compared to 2.64% binding of 125I-CMT protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that C3 was attached to 125I-Epi protein by a covalent bond. Samples eluted from anti-C3-sepharose with hydroxylamine revealed a single, major, 72 kD band, suggesting that C3b attaches almost exclusively to the 72 kD glycoprotein of Epi by a hydroxylamine-susceptible ester bond. An antiserum was prepared from lysates of serum-treated Epi that had been affinity-purified on anti-C3-sepharose. This antiserum immunoprecipitated a single 72 kD component (gp72) from surface-iodinated Epi, and specifically recognized only gp72 from Epi in immunoblots. In contrast to the results with Epi, gp72 on CMT was not found to be an efficient acceptor molecule for C3 deposition. The results are the first to evaluate the acceptor site for C3 deposition on a parasite, and they show that gp72 on Epi, but not gp72 on CMT, serves as the preferential acceptor for C3 during antibody-independent alternative complement pathway activation.
Lack of gene rearrangement and mRNA expression of the beta chain of the T cell receptor in spontaneous rat large granular lymphocyte leukemia lines.
Authors: Reynolds C W , Bonyhadi M , Herberman R B , Young H A , Hedrick S M
Published: June 2004

Using the murine cDNA clone for the beta chain of the T cell antigen receptor, we have examined four highly cytotoxic rat large granular lymphocyte (LGL) leukemia lines for the expression of unique rearrangements and mRNA transcription of the genes coding for the T cell antigen receptor. In contrast to normal rat T cells and nine rat T cell lines, the LGL leukemia lines exhibited no detectable gene rearrangements in the beta chain locus after digestion of LGL DNA by four restriction enzymes. Northern blots containing RNA from these LGL tumor lines demonstrated a low level of aberrant or nonrearranged beta chain transcription (less than 10 copies per cell) but virtually no translatable 1.3 kilobase message. These results demonstrate that LGL leukemia lines which mediate both natural killer (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) activities do not express the beta chain of the T cell receptor. The nature of the NK cell receptor for antigen remains elusive.
Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells.
Authors: Beutler B , Mahoney J , Le Trang N , Pekala P , Cerami A
Published: June 2004

Previous studies have indicated that endotoxin and other bacterial and protozoal products can stimulate macrophages to produce a factor that can suppress the activity of the enzyme lipoprotein lipase (LPL), in vivo and in vitro. In the present report we describe the purification of this factor, cachectin, to apparent homogeneity from the conditioned medium of endotoxin-stimulated RAW 264.7 cells. The isolated protein has an isoelectric point of 4.7 and a subunit molecular weight of 17,000. Although cachectin's isoelectric point and molecular weight are similar to those described for interleukin 1, pure cachectin has no leukocyte-activating factor (LAF) activity. Cachectin at a concentration of 10(-11) M has the ability to suppress the LPL activity of the 3T3-L1 adipocyte cell line by 80%. Binding studies using radio-labeled cachectin and 3T3-L1 adipocytes and C2 myotubules revealed approximately 10(4) high-affinity receptors per cell on both cell types (Ka, 3 X 10(9]. Cachectin receptors were also present on liver membranes but were absent on erythrocytes and lymphocytes. The isolation of cachectin and characterization of its receptor should facilitate further investigations into the role of cachectin and other macrophage mediators in the metabolic derangements that occur during infection and cachexia.
Regulation of IgG memory responses by helper and suppressor T cells activated by the type 2 antigen, polyvinylpyrrolidone.
Authors: Braley-Mullen H
Published: June 2004

T cells from CAF1 mice immunized with various amounts of the type 2 antigen polyvinylpyrrolidone (PVP) were assessed for their ability to provide help to PVP-specific memory B cells for the production of IgG. Low doses (0.0025 micrograms) of PVP consistently activated helper T cells (Th), which were required for the production of IgG by primed B cells. In contrast, T cells from mice primed with higher amounts (0.25 or 25 micrograms) of PVP did not provide significant help to the same B cells for IgG production. Moreover, when mixed with B cells and low-dose PVP-primed Th, T cells from mice primed with 0.25 or 25 micrograms PVP suppressed PVP-specific IgG, but not IgM antibody responses. The suppressor cells induced by higher amounts of PVP were eliminated either by injecting cyclophosphamide (CY) before priming with PVP, or by treating the primed T cells with anti-Lyt-2.2 and C before transfer. Pretreatment of suppressor T cell (Ts) donors with CY or removal of Lyt-2+ T cells not only eliminated Ts activity, but also unmasked significant Th activity in the T cells from high-dose PVP-primed mice. Thus, both low and high amounts of PVP can activate Th, although high amounts of PVP also induce Ts, the activity of which predominates in a normal unfractionated T cell population. The amount of PVP (0.0025 micrograms) that induces dominant help for IgG memory responses was only marginally immunogenic for induction of primary PVP-specific IgM responses, while 0.25 and 25 micrograms PVP, which induce dominant suppression for IgG memory responses, are optimally immunogenic for primary IgM responses. These results are discussed in the context of the inability of most type 2 antigens to elicit primary IgG responses or to prime memory B cells for production of IgG, responses which are dependent on the function of antigen-specific Th.
Interleukin 2 upregulates expression of its receptor on a T cell clone.
Authors: Malek T R , Ashwell J D
Published: June 2004

Stimulation of a class II-restricted, antigen-specific T cell clone with interleukin 2 (IL-2) resulted in substantial increases in both cell surface IL-2 receptor (IL-2-R) and cytoplasmic IL-2-R messenger RNA (mRNA), whereas no increase was observed for cell-surface expression of Thy-1 and L3T4 antigens, and only a modest increase in Thy-1 mRNA was observed. These experiments demonstrate that, after initial acquisition of the IL-2-R, IL-2 as well as antigen is able to directly upregulate both the level of IL-2-R mRNA and cell surface IL-2-R molecules.
Monocyte receptors for fibronectin characterized by a monoclonal antibody that interferes with receptor activity.
Authors: Hosein B , Bianco C
Published: June 2004

We describe a molecule on the surface of human peripheral blood monocytes that appears to be a plasma membrane receptor for fibronectin. We have identified this protein using a monoclonal antibody, A6F10, which prevents the interaction between monocytes and substrate-bound fibronectin. Thus, at least functionally, the antibody appears to recognize the plasma membrane receptor for fibronectin. The antibody and its Fab fragments bound to the cell surfaces of human monocytes, tissue macrophages, and, to a lesser extent, neutrophils. It did not react with fibroblasts, lymphocytes, platelets, or erythrocytes. It bound human and guinea pig cells but did not react with rat, mouse, or hamster cells. In Western blots, this monoclonal antibody bound specifically to a polypeptide with apparent molecular weight of 110,000 and made of a single chain. The antigen recognized by A6F10 was susceptible to trypsin digestion. These observations suggest that the monoclonal antibody A6F10 is directed to the fibronectin receptor of human monocytes.
Distinct recognition phenotypes exist for T cell clones specific for small peptide regions of proteins. Implications for the mechanisms underlying major histocompatibility complex-restricted antigen recognition and clonal deletion models of immune response gene defects.
Authors: Shastri N , Oki A , Miller A , Sercarz E E
Published: June 2004

Using synthetic peptides as antigens, it was found that T cell clones of a given haplotype specific for 13-16 amino acid peptides could be clearly distinguished by the varied influence of amino acid substitutions on recognition. This was true for different antigenic determinants within peptides 81-96 and 74-86 of hen egg-white lysozyme, recognized in the context of the I-Ab and I-Ak molecules, respectively. Considerable complexity was demonstrated in the induced T cell repertoire specific for apparently single determinants, which implies that diversity of T cell recognition approaches that for B cells. The implications of the degeneracy of T cell recognition are discussed in the context of mechanisms through which Ia molecules restrict recognition and theories of Ir gene defects.
In vivo activation of macrophage C3 receptors for phagocytosis.
Authors: Griffin F M , Mullinax P J
Published: June 2004

We assessed the effects of exposure to immune complexes in vivo on macrophages' Fc receptor function and C3 receptor function. Peritoneal macrophages from mice injected intraperitoneally with immune complexes were markedly impaired in their ability to phagocytize via their Fc receptors but had acquired the ability to phagocytize via their C3 receptors. In vivo activation of macrophages' C3 receptors for phagocytosis required T lymphocytes, because macrophages from athymic mice could not be activated by injection of immune complexes. The requirement for both T lymphocytes and immune complexes for activation of macrophages' C3 receptors in vivo is identical to the requirements for activation of macrophages' C3 receptors in vitro, suggesting that the mechanisms we have identified for activation of these receptors in vitro are the same mechanisms by which the receptors are activated for phagocytosis in vivo. The susceptibility of macrophages' Fc receptors to blockade by immune complexes and the activation of their C3 receptors for phagocytosis in a milieu containing immune complexes suggest that it may be macrophages' C3 receptors, not their Fc receptors, that are primarily responsible for promoting phagocytosis of opsonized microorganisms in immune hosts.
Rapid polymerization of Entamoeba histolytica actin induced by interaction with target cells.
Authors: Bailey G B , Day D B , Gasque J W
Published: June 2004

Within 5 s of challenge of Entamoeba histolytica trophozoites with red blood cells (RBC), attachment and deformation of target cells occurred at multiple sites on the amoeba surface. Many trophozoite-target interfaces were outlined with a ring of polymerized amoeba actin, revealed by rhodamine-phalloidin staining of glutaraldehyde-fixed and Triton-X 100-extracted cells. The beginnings of phagocytic pseudopods rimmed many targets. The phagocytic membrane and underlying actin network grew uniformly about a target cell, which became dramatically elongated and constricted, sometimes severed, as it entered the amoeba. Total engulfment of RBC targets occurred within 10 s. By methanol extraction and spectrofluorimetric measurement of bound rhodamine-phalloidin we were able to quantitate polymerized actin in amoebae. Interaction with target cells was accompanied by a net increase of up to twofold in the average polymerized actin content of trophozoites. This reached a maximum during the period of most active phagocytosis (4 min after challenge at 25 degrees C), and declined as phagocytic activity diminished (8-16 min). Challenge with latex beads of similar size and number, which E. histolytica phagocytized more slowly than RBC, induced neither a detectable increase in polymerized actin content nor appearance of polymerized actin at the contact interface. RBC inhibited phagocytosis of latex beads, but the reverse did not occur. The results demonstrate a rapid, recognition-specific stimulation of reorganization of the actin cytoskeleton of E. histolytica induced by binding to target cells. Vigorous phagocytic activity is frequently an immediate consequence of cell-cell contact, which emphasizes the importance of this process in the contact-mediated attack mechanism of this pathogen. The quantitative assay of polymerized actin may be useful in further studies of this mechanism.
Collagen-induced arthritis in mice. Localization of an arthritogenic determinant to a fragment of the type II collagen molecule.
Authors: Terato K , Hasty K A , Cremer M A , Stuart J M , Townes A S , Kang A H
Published: June 2004

Purified chick type II collagen was cleaved with cyanogen bromide (CB), and the resulting peptides isolated and renatured. Sera from arthritic DBA/1 mice, immunized with chick type II collagen, were tested for reactivity with each peptide. The sera preferentially recognized peptides 11, 10, and 8, in that order. Some reactivity was also detected to peptides 9, 7, and 12. Because arthritis depends upon binding of antibody to autologous type II collagen in the joint, sera were also tested for reactivity with mouse type II collagen. There was a strong positive correlation between reactivity with peptide 11 and reactivity with mouse collagen, but no correlation was found with any of the other peptides. Peptides 11, 10, and 8 were also used for immunization. Antibodies were detected in response to each of these peptides, but arthritis developed only in mice immunized with peptide 11. We conclude that a major immunogenic and arthritogenic epitope on type II collagen resides in the region of the molecule represented by CB peptide 11.
Unequal sister chromatid exchange. A mechanism affecting Ig gene arrangement and expression.
Authors: Tilley S A , Birshtein B K
Published: June 2004

Two gamma 2a gene forms in the MPC11 mouse myeloma cell line (gamma 2b, K) have been localized to the expressed H chain chromosome, where they exist in tandem downstream of the expressed gamma 2b gene. The gamma 2a gene duplication has apparently occurred by spontaneous, unequal sister chromatid exchange (SCE) in a B lymphoid precursor of MPC11 or in MPC11 itself. It is especially significant that either of the tandem gamma 2a gene forms may be used in the class switch from gamma 2b to gamma 2a production occurring in the MPC11 line, indicating that heavy chain constant region (CH) gene duplication (or deletion) may be a mechanism normally impinging on CH gene expression. The gamma 2a genes of MPC11 undergo further copy number variation via unequal SCE in variants of this line derived by mutagenesis and selection for altered H chain production. The remarkable frequency with which copy number variation occurs in these cells suggests that unequal SCE may play a physiological role in CH gene arrangement and expression.
Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell-deficient W/Wv mice. Evidence that cultured mast cells can give rise to both connective tissue type and mucosal mast cells.
Authors: Nakano T , Sonoda T , Hayashi C , Yamatodani A , Kanayama Y , Yamamura T , Asai H , Yonezawa T , Kitamura Y , Galli S J
Published: June 2004

Both connective tissue mast cells and mast cells grown in vitro are derived from multipotential hematopoietic stem cells, but these two mast cell populations exhibit many differences in morphology, biochemistry, and function. We investigated whether the phenotype of cultured mast cells or their progeny was altered when the cells were transferred into different locations in vivo. Cultured mast cells were immature by ultrastructure, and stained with alcian blue but with neither safranin or berberine sulfate, a fluorescent dye that binds to the heparin of connective tissue mast cell granules. By contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice 10 wk after intraperitoneal injection of cultured WBB6F1-+/+ or C57BL/6-bgJ/bgJ mast cells stained with both safranin and berberine sulfate. Staining with berberine sulfate was prevented by treatment of the cells with heparinase but not chondroitinase ABC, suggesting that the adoptively transferred mast cell population had acquired the ability to synthesize and store heparin. Furthermore, the recovered mast cells were indistinguishable by ultrastructure from the normal mature peritoneal mast cells of WBB6F1-+/+ mice, and contained substantially more histamine than mast cells studied directly from culture. Intravenous injection of cultured mast cells resulted in the development of safranin-and berberine sulfate-positive mast cells in the peritoneal cavity, spleen, skin, and glandular stomach muscularis propria. Mast cells also developed on the glandular stomach mucosa, but these cells stained with alcian blue rather than safranin, and did not stain with berberine sulfate. This result suggests that cultured mast cells can give rise to mast cells of either the connective tissue type or mucosal phenotype, depending on anatomical location. Furthermore, transplantation of cultured mast cells into WBB6F1-W/Wv mice had no measurable effect on the anemia of the recipient mice, suggesting a possible strategy for repairing the mast cell deficiency of WBB6F1-W/Wv mice without affecting other bone marrow-derived populations such as erythrocytes. Intravenous injection of representative connective tissue type mast cells (30-50% pure peritoneal mast cells derived from WBB6F1-+/+ mice) gave results similar to those obtained with cultured mast cells: mast cells developing in the peritoneal cavity, skin, spleen, and glandular stomach muscularis propria of WBB6F1-W/Wv recipients stained with safranin and berberine sulfate, whereas mast cells developing in the mucosa of the glandular stomach stained only with alcian blue.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigen-specific, I-A-restricted suppressor hybridomas with spontaneous cytolytic activity. Functional properties and lack of rearrangement of the T cell receptor beta chain genes.
Authors: Blanckmeister C A , Yamamoto K , Davis M M , Hämmerling G J
Published: June 2004

T suppressor (Ts) hybridomas were produced by fusion of the III/4 T cell hybridoma with splenic T cells from CBA mice tolerized with subimmunogenic doses of bovine serum albumin (BSA). Both the Ts hybridoma cells and a suppressor factor (TsF) inhibited in an antigen-specific and I-Ak-restricted fashion the in vitro proliferative response of BSA-immunized lymph node cells. In addition to the suppressive activity, the hybridoma lines displayed spontaneous cytotoxicity against various tumor targets. The isolation of Ts subclones that are suppressive but not cytolytic, as well as the existence of the noncytolytic TsF, indicates that suppression of antigen-specific T cell proliferation is not dependent on cytolytic activity. The Ts hybridomas were I-A restricted, as are many T helper cells. Therefore, a potential similarity with respect to antigen receptor genes was expected. Southern blot analysis with a probe specific for genes encoding the beta chain of the T cell receptor on T helper and T killer cells revealed no rearrangement of the beta genes in the Ts cells. The data imply that neither the antigen receptor on the I-A-restricted Ts cells nor the receptor involved in the cytolytic interaction with tumor targets use the same beta chain constant region as T helper and T killer cells.
Isolation and characterization of resident stromal macrophages and hematopoietic cell clusters from mouse bone marrow.
Authors: Crocker P R , Gordon S
Published: June 2004

In situ studies with the mouse macrophage (M phi)-specific antibody, F4/80, have shown that resident M phi in femoral bone marrow (RBMM) form hematopoietic islands with immature myelomonocytic and erythroid cells (Hume, D. A., et al. 1983. J. Exp. Med. 158: 1522). We have isolated these islands (clusters) by collagenase digestion, purified them from single cells by velocity sedimentation, and analyzed their cellular content. The clusters, ranging from 5- to 100 cells, constituted approximately 7% of the total nucleated cells, and greater than 70% contained at least one strongly staining, F4/80+ central M phi. In comparison, less than 26% showed reactivity for alkaline phosphatase, a marker of fibroblastoid reticulum cells. Compared with the nonclustering population, clusters were enriched with RBMM, fibroblastoid cells, and immature hematopoietic cells, but depleted of mature granulocytes and erythrocytes. The RBMM population was purified from other cells in clusters by selective adherence to glass and was compared with resident peritoneal M phi (RPM) for morphology and the presence of antigens, receptors, and enzymes. RBMM spread more extensively than RPM and frequently extended delicate plasma membrane processes. These and subsequent differences were not attributable to the collagenase treatment. Both M phi populations stained positively with antibodies F4/80 and 2.4G2 (Fc receptor IgG1/2b), bore mannosyl/fucosyl receptors, and showed reactivity for acid phosphatase and nonspecific esterase I. In contrast to RPM, RBMM had no detectable Mac-1 antigen (CR3) or complement receptors, but bore higher levels of Fc receptors (IgG2a and IgG2b) and Ia antigens. In addition, RBMM possessed a novel hemagglutinin activity for unopsonized sheep erythrocytes, which was not present on RPM. RBMM showed no respiratory burst activity in response to zymosan particles, but ingested them avidly. The growth properties of clustering and nonclustered populations were compared by measurement of [3H]thymidine incorporation and progenitor assays. Cells in clusters incorporated three- to fourfold more thymidine than nonclustered cells even in the absence of exogenous growth factors, and autoradiography demonstrated that RBMM made contact with proliferating cells. In contrast, the clusters contained over threefold fewer granulocyte/M phi progenitors compared with nonclustering cells. When clusters were cultivated for up to 3 d, there was rapid outgrowth of monocytes and fibroblastoid cells. These studies demonstrate that RBMM bear a distinct morphology and phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of the growth and functions of cloned murine large granular lymphocyte lines by resident macrophages.
Authors: Minato N , Amagai T , Yodoi J , Diamanstein T , Kano S
Published: June 2004

Using cloned lines with the morphology of large granular lymphocytes (LGL) from BALB/c mice, we studied the exact requirements for proliferation and their functional characteristics, as well as their regulation. Although these cloned LGL lines were interleukin 2 (IL-2) dependent for growth, experiments using human recombinant IL-2 (rIL-2), known to be active on murine cells, indicated that IL-2 was a necessary but not sufficient factor. Coexistance of normal macrophages in addition to rIL-2 was found to support continuous proliferation of cloned LGL in vitro. This role of macrophages could be replaced by partially purified IL-1 derived from macrophage-conditioned medium. An IL-2 binding assay using 125I-rIL-2 suggested that the role of normal macrophages was to selectively induce and/or maintain high affinity IL-2 receptors (IL-2R) (Kd, 0.2-0.5 nM) without affecting low affinity ones (Kd, 10-30 nM). Functional studies indicated that most of the LGL clones killed various combinations of representative groups of natural killer (NK)-susceptible target cells, including leukemic cells (YAC-1, RL male 1), virus-infected cells (HeLa-measles, HeLa-herpes simplex virus), and normal bone marrow cells (BMC), whereas none of them affected any of NK-resistant target cells, including uninfected HeLa cells. Some of these clones also suppressed in vitro hematopoiesis. Such characteristic cytotoxic spectra, as well as serological phenotypes (Thy-1+, Lyt-1-2-, asialo GM1-positive, T200+, TdT-, Fc receptor-positive) indicated that these LGL clones exactly represent endogenous NK cells, rather than a variety of anomalous killer cells generated in various culture conditions. Although there was significant heterogeneity of cytotoxic spectrum among LGL clones, no clonotypic distribution of specificities was observed. Normal macrophages were found to modulate the functional expression of LGL clones. They augmented the cytotoxic potential of the clones against leukemic and virus-infected targets, but suppressed intrinsic reactivity against normal BMC. Similarly, LGL clones maintained with macrophages showed much less suppressive effect on in vitro hematopoiesis. The present observations on the interaction of cloned LGL and normal macrophages provide a basic explanation for the mechanisms by which the immediate responsiveness to IL-2 of the NK effector system, without exogenous stimulation, and the functional selectivity toward abnormal rather than normal cells, are actively maintained in vivo.