bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of variable region gene expression and environmental selection in determining the antiphosphorylcholine B cell repertoire.
Authors: Klinman N R , Stone M R
Published: June 2004

To evaluate the role of environmental selective processes, as opposed to variable region gene expression, in the determination of B cell repertoire expression, we have assessed the phosphorylcholine (PC)-specific repertoire of precursor cells that remain in bone marrow cell populations after the removal of surface immunoglobulin (sIg)-bearing cells. Such cells are assumed to represent a stage in B cell maturation before the expression of sIg, and thus at a time when they have not as yet interfaced with environmental influences that operate through sIg receptors such as antigenic stimulation, tolerance, or antiidiotypic regulation. The repertoire as expressed in these cells, therefore, should reflect the readout of immunoglobulin variable region genes as they are expressed in progenitors to B cells. The results of these studies indicate that, as in mature primary B cell pools of BALB/c mice, the majority of PC-responsive sIg- bone marrow cells are of the T15 clonotype. Thus, environmental selective mechanisms would not appear to be required for the high frequency of B cells of the T15 idiotype in the primary B cell repertoire of BALB/c mice. Analysis of the sIg- bone marrow cells in (CBA/N X BALB/c)F1 male mice demonstrated that the deficit of PC-responsive mature B cells, which is a characteristic of this murine strain, must occur after receptor expression, since a normal frequency of PC-responsive and T15-expressing cells is present in their sIg- bone marrow population. Finally, these same mice were used to obtain bone marrow cell preparations from individual leg bones, so as to permit an analysis of the occurrence of T15+ and T15- clonotypes within individual bone marrow populations. The findings from these studies indicate that T15+ B cells occur as a high frequency event within bone marrow generative cell pools. Furthermore, bone marrow populations that are positive for PC-responsive precursor cells often display multiple copies of such precursor cells that are exclusively either T15+ or T15-. This finding indicates that clonal expansion of cells within the B cell lineage apparently occurs before immunoglobulin receptor acquisition.
Identification of a new component in the murine Ia molecular complex.
Authors: Sant A J , Schwartz B D , Cullen S E
Published: June 2004

In this report, we describe a previously unidentified component in the murine Ia antigen complex. SDS-PAGE analysis of anti-Ia immunoprecipitates prepared from spleen cells biosynthetically labeled with 35S-sulfate showed no detectable incorporation of 35SO4 into alpha, beta, or Ii chains but did not reveal the presence of a novel sulfate-bearing molecule of considerable molecular weight heterogeneity (46-69-kdaltons). The 46-69-kdalton molecule could be precipitated with monoclonal antibodies specific for I-A, I-E, and Ii glycoproteins but was not seen in control precipitates, nor in association with IgG or class I MHC molecules. Preliminary biochemical characterization indicated that the 46-69-kdalton product is extremely polydisperse, both in charge and apparent molecular weight, is sensitive to proteases, and bears the sulfate moiety on a large pronase-resistant structure. These results suggested this component might be a proteoglycan. Definitive identification of this component as a proteoglycan was accomplished by selective enzymatic degradation experiments which showed that the sulfate-bearing component of the 46-69-kdalton molecule is chondroitin 6-sulfate.
Ultrastructural studies of human lymphoid cells. mu and J chain expression as a function of B cell differentiation.
Authors: Hajdu I , Moldoveanu Z , Cooper M D , Mestecky J
Published: June 2004

J chain expression was examined as a function of the stage in differentiation along the B cell axis in humans. Intracellular distribution of J and mu chains in leukemic HLA-DR+ null and pre-B cells, and in normal B cells stimulated with pokeweed mitogen (PWM) was determined by immunoelectron microscopy and radioimmunoassay (RIA). J chain was detected in leukemic null and pre-B cells on free and membrane-bound ribosomes in the cytoplasm, or on perinuclear cisternae. Mu chain was found on free ribosomes and ribosomal clusters in leukemic pre-B cells but was absent in the leukemic null cells. In pre-B cell lines, mu chain was seen within rough endoplasmic reticulum (RER) and the Golgi apparatus whereas J chain was not detected in these organelles. However, both mu and J chain were detected in RER and the Golgi apparatus of immature and mature plasma cells induced by PWM stimulation of normal peripheral blood lymphocytes. Low levels of J chain were also detected by RIA in lysates of leukemic null and pre-B cells. Most of the intracellular J chain became detectable after reduction and alkylation of cell lysates, and free J chain was not found in the culture supernatants. The amount of intracellular and secreted immunoglobulin-bound J chain increased dramatically after PWM stimulation of peripheral blood lymphocytes. The majority of J chain-positive cells seen over an 8 d culture interval were lymphocytes and lymphoblasts, while mu chain was found primarily in plasma cells. These results suggest that J chain expression precedes mu chain synthesis during B cell differentiation and that a combination of the two chains for secretion is not initiated until the onset of plasma cells maturation.
Identification of a specific interleukin 1 inhibitor in the urine of febrile patients.
Authors: Liao Z , Grimshaw R S , Rosenstreich D L
Published: June 2004

The urine of febrile patients has been found to contain high concentrations of an inhibitor of interleukin 1 (IL-1)-induced thymocyte proliferation. The inhibitor is specific for IL-1 and does not block the effects of interleukin 2 (IL-2) or phytohemagglutin (PHA) on thymocytes, and it is not nonspecifically toxic for these cells. IL-1 inhibitor can be found in the urine of normal individuals and afebrile patients, but is present in increased concentrations in the urine of patients with fever of diverse etiologies. Preliminary physicochemical characterization indicates that the inhibitor is a 20-40-kdalton protein.
Studies on the fibronectin receptors of human peripheral blood leukocytes. Morphologic and functional characterization.
Authors: Pommier C G , O'Shea J , Chused T , Yancey K , Frank M M , Takahashi T , Brown E J
Published: June 2004

We have investigated the interactions between plasma fibronectin (Fn) and human peripheral blood phagocytic cells. As shown by studies of the binding of Fn-coated fluorescent microspheres (Fn-ms), both polymorphonuclear leukocytes (PMN) and monocytes had specific binding sites for Fn at the plasma membrane. However, as purified from blood, only monocytes were stimulated by Fn to become more actively phagocytic. This increase in phagocytosis was reflected by an Fn-induced increase in the ingestion of IgG-coated erythrocytes and, more dramatically by an Fn-dependent initiation of phagocytosis of C3b-coated erythrocytes. Despite this difference between PMN and monocytes in the functional consequences of Fn binding, the cell surface molecules responsible for Fn binding on the two cell types shared many characteristics. On both cells, binding of Fn-ms was inhibited by sufficient concentrations of fluid-phase Fn; both PMN and monocytes bound fewer Fn-ms at 4 degrees C than at 37 degrees C; both achieved maximal binding at similar Fn-ms/cell ratios; and phenylmethylsulfonyl fluoride did not inhibit Fn-ms binding to either cell type. Most dramatically, monoclonal anti-Fn antibodies that inhibited binding of Fn-ms to one cell type inhibited binding to both; conversely, monoclonal anti-Fn antibodies that did not inhibit Fn-ms binding to either cell type did not inhibit binding to the other. Fn will stimulate PMN to a more actively phagocytic state, like that induced in monocytes, if the PMN are first exposed to C5a or N-formyl-methionyl-leucylphenylalanine. This effect occurs without apparent change in the number of Fn receptors. We conclude that the PMN and monocyte receptors for Fn are very similar, but that their milieu is very different in the two cells as purified from peripheral blood. Whereas Fn induces increased phagocytosis in monocytes, PMN must be activated before the Fn can be effective.
Serologic cross-reactivity between Class I MHC molecules and an H-2-linked differentiation antigen as detected by monoclonal antibodies.
Authors: Sharrow S O , Flaherty L , Sachs D H
Published: June 2004

Analysis of anti-Class I major histocompatibility complex (MHC) monoclonal antibodies by immunofluorescence and flow microfluorometry demonstrated an unexpected cross-reactivity. Two of fifteen antibodies examined (20-8-4, anti-Kb,Kd,r,s and 34-1-2, antiKd,Dd,Kb,r,s,q,p) were observed to detect an antigen determined by gene(s) mapping to the right of H-2D. Two-color immunofluorescence analysis demonstrated that this antigen, unlike classical H-2K and D antigens, was expressed in high amounts on peripheral T cells, but only weakly on Ia-positive cells and on small subpopulations of thymus and bone marrow cells. Mapping, absorption, blocking, and tissue distribution studies suggested that the cross-reactive antigen is Qa-like, but distinct from previously described Qa antigens. Thus, these data demonstrate serological cross-reactivity between a Class I MHC antigen and a differentiation antigen determined by genes linked to H-2. It seems likely that the gene responsible for this new antigen is one of the numerous Class I-like sequences detected by DNA hybridization analyses, but previously undefined in terms of tissue expression. These data suggest that many of these DNA sequences may be expressed in specific tissues and that cross-reactions of anti-Class I MAbs may provide useful probes for studying the products of such homologous genes.
Local opsonization by secreted macrophage complement components. Role of receptors for complement in uptake of zymosan.
Authors: Ezekowitz R A , Sim R B , Hill M , Gordon S
Published: June 2004

We have examined the role of macrophage (M phi plasma membrane receptors for the cleaved third complement component (iC3b; CR3) and mannosyl, fucosyl terminated glycoproteins (MFR) in uptake of unopsonized zymosan. Monoclonal antibodies against CR3, M1/70 (Mac-1) and MO1, each inhibited approximately 50% of uptake of 125I-zymosan by murine and human M phi, respectively. Yeast mannan inhibited 0-50% of zymosan uptake in various M phi, in parallel with their expression of MFR. We demonstrated that M phi were the source of C3 in our assay and that the activity of other components of the complement system, namely a C3 convertase, factor I, and a factor I cofactor were also present in serum-free cultures of human monocytes. Macrophage C3 was deposited rapidly, within 10 min, on the zymosan particles and mediated binding, ingestion, and stimulation of superoxide release in BCG-activated and thioglycollate-elicited peritoneal M phi via CR3. Local secretion of complement proteins by M phi themselves can therefore opsonize pathogens and cells able to activate the alternative pathway and effect their destruction.
Subcellular localization of the PGE2 synthesis activity in mouse resident peritoneal macrophages.
Authors: Darte C , Beaufay H
Published: June 2004

The aim of this work was to establish, on a quantitative basis, the subcellular distribution of the enzyme system that converts arachidonic acid into prostaglandin (PG) E2 in mouse resident peritoneal (MRP) macrophages. Kinetic studies were conducted on cell-free extracts derived from cells cultivated for 1 d, using [1-14C]arachidonic acid as substrate and measuring the label in PGE2 after extraction and thin layer chromatography. The activity was synergistically enhanced by L-adrenaline and reduced glutathione, inhibited by indomethacin, and linearly related to the concentration of the cell-free extract. It was labile at 0 degrees C in the medium used for homogenization and fractionation of the cells (half-life less than 2 h). Addition of catalase (0.15 mg/ml) to the suspension medium increased the initial activity (by congruent to 70%) and the stability (half-life congruent to 6 h) of the enzyme in cytoplasmic extracts. It enabled us to establish the density distribution after isopycnic centrifugation in a linear gradient of sucrose. The sample centrifuged consisted of untreated cytoplasmic extracts, or cytoplasmic extracts treated with digitonin and Na pyrophosphate. Comparison of the centrifugation behavior of PGE2 synthesis activity with that of various enzymes used as reference for the major subcellular entities has revealed that PGE2 synthesis fairly fits the density profile of sulfatase C in each case. The conclusion is that at least the rate-limiting reaction in the conversion of arachidonic acid into PGE2 is catalyzed by an enzyme associated with the endoplasmic reticulum.
The HLA-DR4 family of haplotypes consists of series of distinct DR and DS molecules.
Authors: Nepom G T , Nepom B S , Antonelli P , Mickelson E , Silver J , Goyert S M , Hansen J A
Published: June 2004

Among DR4-associated HLA-D antigens, distinct and consistent structural variations were found for the products of two human "Ia-like" loci, DR and DS. Analysis of neuraminidase-treated immunoprecipitated DR molecules from 15 HLA-DR4-associated HLA-D homozygous B-lymphoblastoid cell lines by two dimensional polyacrylamide gel electrophoresis identified five distinct DR beta chains. In addition, gel analysis of immunoprecipitated DS molecules identified three distinct DS beta chains. Altogether, five distinct DR4 haplotypes were defined according to the observed structural diversity of the DR and DS beta chains. These gene products presumably contribute the dominant polymorphisms recognized by T cells in mixed lymphocyte reaction (MLR). Thus, these studies indicate that the serologic specificity known as HLA-DR4 is not a single haplotype, but a determinant present on products of individual loci arrayed into distinctly different haplotypes. These findings suggest that distinct products of individual loci, rather than conventional HLA specificities defined by alloimmune sera, may represent the genetic markers relevant to HLA-D/DR associated diseases.
Activation specificity of arsonate-reactive T cell clones. Structural requirements for hapten recognition and comparison with monoclonal antibodies.
Authors: Rao A , Faas S J , Cantor H
Published: June 2004

We describe clones of hapten-specific inducer T cells from (BALB/c X A/J)F1 mice that respond to the p-azobenzenearsonate hapten conjugated to carrier proteins or directly conjugated to antigen-presenting cells. Some of the clones are also activated by haptens structurally related to arsonate. All activating analogues are recognized by each clone in association with the same major histocompatibility complex (MHC) protein as is arsonate. Weakly activating and nonactivating analogues are immunogenic in D2.GD amd (BALB/c X A/J)F1 mice, since they can effectively activate primed lymph node cells or long-term hapten-reactive cell lines. Hence the specificities of these clones may reflect their intrinsic recognition of arsonate and its analogues, rather than more efficient presentation of certain analogues than of others by antigen-presenting cells, or differential recognition of associated MHC epitopes by the clones. We compare the activation specificities of the clones with the binding specificities of monoclonal antibodies to arsonate, and discuss structural features of the analogues that may be important for activation and binding. Our results suggest that a site (or subsite) on arsonate-reactive T cell clones may interact directly with hapten, and may be experimentally separable from the site (or subsite) for MHC determinants.
Rabbit and rat C-reactive proteins bind apolipoprotein B-containing lipoproteins.
Authors: Rowe I F , Soutar A K , Trayner I M , Baltz M L , de Beer F C , Walker L , Bowyer D , Herbert J , Feinstein A , Pepys M B
Published: June 2004

Immobilized rabbit and rat C-reactive protein (CRP) were found to selectively bind apolipoprotein B (apoB)-containing lipoproteins (low density lipoprotein, LDL and very low density lipoprotein, VLDL) from whole serum in a manner similar to that previously reported with human CRP. In acute phase human serum the CRP is in a free form, not complexed with lipoprotein or any other macromolecular ligand, and in acute phase serum from most rabbits fed on a normal diet the rabbit CRP was also free. However, in acute phase serum or heparinized plasma from hypercholesterolemic rabbits part or all of the CRP was found by gel filtration and immunoelectrophoretic techniques to be complexed with beta-VLDL, an abnormal apoB-containing plasma lipoprotein present in these animals. The presence of extent in different serum samples of CRP complexed with lipoprotein correlated closely with the serum apoB concentration. The formation of complexes between native, unaggregated rabbit CRP in solution and apoB-containing lipoproteins was readily demonstrable experimentally both with the isolated proteins and in whole serum. In all cases these interactions were calcium-dependent and inhibitable by free phosphoryl choline. The present findings extend earlier work in man and the rabbit and indicate that among the C-reactive proteins from different species, which are structurally highly conserved, the capacity for selective binding to apoB-containing plasma lipoproteins is also a constant feature. These interactions may therefore be related to the in vivo function of CRP in all species and this function may in turn be relevant to pathological conditions, such as atherosclerosis, in which lipoproteins are important.
Regulatory idiotopes. Induction of idiotype-recognizing helper T cells by free light and heavy chains.
Authors: McNamara M , Kohler H
Published: June 2004

Previously, we have demonstrated the induction of T helper cells that recognize idiotype by antigen (19), idiotype (20), and antiidiotype (12). The T cell population has been characterized and found to recognize both the T15 and M167 myeloma proteins, which share PC binding specificity but differ in idiotypic specificities. In the present work, we used isolated heavy and light chains of T15 and M167 to generate T helper cells, and examined the response to trinitrophenyl (TNP)-T15 and TNP-M167. We found that the heavy chains induced a dose-dependent response to TNP-T15 and TNP-M167, while the light chain priming was ineffective. When isolated chains of a monoclonal anti-T15 antibody (F6-3) were used to induce idiotype-recognizing T cells, only the F6-3 light chains generated T cell help for TNP-T15 and TNP-M167. Evidently, the idiotypic determinant that is recognized by the T cells is not dependent upon the conformation of combined heavy and light chains. These data show that the Th2 helper cells for the T15/M167 idiotopes are induced by free heavy chains of T15 and M167; the Th1 T cells that interact with the Th2 population, of T15 and M167; the Th1 T cells that interact with the Th2 population, however, can be triggered by free light chains of an antiidiotypic hybridoma antibody. These provocative findings suggest a new model for the T helper cell network.
Sequence of a cDNA encoding Basilea kappa light chains (K2 isotype) suggests a possible relationship of protein structure to limited expression.
Authors: Bernstein K E , Lamoyi E , McCartney-Francis N , Mage R G
Published: June 2004

We present the complete sequence of a cDNA encoding rabbit immunoglobulin kappa light chains of the Basilea isotype (K2). Although all rabbits seem to possess a K2 constant region gene, expression of this gene in most rabbits is minimal if present at all. Even in Basilea rabbits the majority of expressed immunoglobulins are of lambda type. We find that the sequence of our Basilea cDNA constant region and the sequence of a "silent" K2 gene from b4 rabbits (bas-N4) are almost identical. The bas (K2) isotype lacks cysteine at position 171 in the constant region that is present in all K1 constant regions and usually forms an interdomain disulfide bond, with a cysteine at position 80 of the variable region. We postulate that one factor contributing to the low expression of the bas (K2) isotype could be a paucity of V kappa regions lacking cysteine at position 80. If a typical rabbit V kappa encoding Cys at position 80 is rearranged and expressed with th K2 isotype. B cells with mRNAs encoding light chains with free sulfhydryl groups would result. These cells may fail to form functional immunoglobulin receptors. Only a small subset of rabbit variable regions that lack the cysteine at position 80 would rearrange and encode K2 light chains lacking a free sulfhydryl group.
Murine tissue amyloid protein AA. NH2-terminal sequence identity with only one of two serum amyloid protein (ApoSAA) gene products.
Authors: Hoffman J S , Ericsson L H , Eriksen N , Walsh K A , Benditt E P
Published: June 2004

Amyloid protein AA is the presumptive fragment of an acute phase serum apolipoprotein, apoSAA. Two major murine apoSAA isotypes (apoSAA1 and apoSAA2) have been identified. The NH2-terminal amino acid sequences of purified murine apoSAA1 and apoSAA2 have been examined and compared with that of murine amyloid protein AA. Our results indicate that apoSAA1 and apoSAA2 are separate gene products and that amyloid protein AA has NH2-terminal amino acid sequence identity with only one of these isotypes, namely apoSAA2.
Creatine kinase expression and creatine phosphate accumulation are developmentally regulated during differentiation of mouse and human monocytes.
Authors: Loike J D , Kozler V F , Silverstein S C
Published: June 2004

We have studied the expression of creatine kinase (CK) and the accumulation of creatine phosphate during the differentiation of human and mouse peripheral blood monocytes. Mouse monocytes cultured for 24 h do not contain detectable levels of CK and creatine phosphate. However, resident tissue macrophages and inflammatory elicited macrophages obtained from the peritoneal cavities of mice have 70 and 300 mU per mg protein of CK activity and contain 3 and 6 mol of creatine phosphate per mol of ATP, respectively. The major isozyme of CK in these cells has been identified as the brain form. These findings suggest that the differentiation of monocytes into macrophages is associated with the expression of CK and the accumulation of creatine phosphate. We have found a similar pattern in human monocytes. Human blood monocytes, maintained in culture for 24 or 48 h, do not contain detectable levels of CK or creatine phosphate. Monocyte-derived macrophages (monocytes maintained in tissue cultures for 1 to 2 wk) have up to 100 mU per mg protein of CK activity and contain 0.5 mol of creatine phosphate per mol of ATP. Human macrophages express multiple isozymes of CK including the brain (BB) and possibly the mitochondrial forms of this enzyme. Thus, the expression of CK and the accumulation of creatine phosphate in human monocytes is induced by their in vitro cultivation. The induction of CK during in vitro cultivation occurs independently of the concentration of creatine in the medium. However, the size of the creatine phosphate pool varies with respect to extracellular creatine concentration. Creatine phosphate and CK are not detectable in freshly isolated human lymphocytes, polymorphonuclear leukocytes or erythrocytes, but are found in freshly isolated human platelets.
Idiotope regulation by isotype switch variants of two monoclonal antiidiotope antibodies.
Authors: Müller C E , Rajewsky K
Published: June 2004

Previous work has shown that the injection of antiidiotope antibodies specific for idiotopes of the germline-encoded anti-(4-hydroxy-3-nitro-phenyl) acetyl (NP) antibody B1-8 enhanced or suppressed the expression of B1-8 idiotopes in subsequent humoral anti-NP responses, depending on the dose and perhaps also the isotype of the injected antibody. To formally answer the question of whether the isotype of an antiidiotope determines its effector function in this type of idiotypic control, we have performed regulatory experiments with isotype switch variants selected from two hybridomas secreting anti-B1-8 idiotopes of CBA (Ighj) and C57BL/6 (Ighb) origin. The antibodies of each variant family differ from each other only in the constant region of the heavy chain. The results show that, irrespective of whether an antiidiotope antibody belongs to the IgG1, IgG2b, IgG2a, or IgE class, a 10-ng dose enhances idiotope expression whereas a dose of 10 micrograms exerts a suppressive effect. It emerges from the present and parallel data that the expression of antibody V regions resembling idiotypically that of antibody B1-8 can be enhanced and suppressed by any of four antiidiotope antibodies that recognize distinct idiotopes on those V regions. This suggests that the initial step in the regulatory process induced by an antiidiotope is its binding to antibody V regions carrying the target idiotope. The antiidiotopes preferentially regulate the expression of antibodies that coexpress with the target idiotope other B1-8 idiotopes, despite the fact that some B1-8 idiotopes are also expressed independently of each other in anti-NP responses of idiotypically unmanipulated mice. This finding may reflect high affinity binding of the antiidiotopes to the target against which they were originally raised (i.e., antibody B1-8) or, more likely, a preferential recognition of B1-8-like V regions by regulatory T cells.
Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin.
Authors: Stone-Wolff D S , Yip Y K , Kelker H C , Le J , Henriksen-Destefano D , Rubin B Y , Rinderknecht E , Aggarwal B B , Vilcek J
Published: June 2004

Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.
Human pulmonary macrophage-derived mucus secretagogue.
Authors: Marom Z , Shelhamer J H , Kaliner M
Published: June 2004

Human pulmonary macrophages (PM) obtained from surgically removed human lung tissue released a factor after exposure to activated zymosan that caused cultured human airways to release increased amounts of radiolabeled mucous glycoproteins. The factor was released maximally after 4-8 h of zymosan exposure and caused a dose-related increase in glycoprotein release; it was termed macrophage-derived mucus secretagogue (MMS). MMS release was produced in a dose-dependent fashion by activated but not by nonactivated zymosan. The activation of zymosan was C3 dependent, and C3b-coated Sepharose was also an effective stimulant. The data suggested that cell surface activation of the PM was a sufficient stimulus to cause MMS release and that both C3-dependent activation as well as Fc receptor activation were effective. The synthesis of MMS was sensitive to cycloheximide, and no active MMS was detectable intracellularly. To determine if MMS might be one of the oxidative derivatives of arachidonic acid, PM were incubated with cyclooxygenase and lipoxygenase inhibitors before activation. These maneuvers did not influence MMS generation. MMS-rich supernatants were then extracted into organic solvents or exposed to lipophilic resin; in both cases, MMS remained in the aqueous phase. Thus, MMS is not a derivative of arachidonic acid. Sequential fractionation of MMS on ultramembrane and gel filtration followed by isoelectric focusing and gel filtration indicated that MMS is a small (approximately 2000 daltons), acidic (pI, 5.15) molecule. Therefore, surface activation of human PM results in the synthesis and release of a small acidic molecule that causes airway mucous glands to secrete increased quantities of mucous glycoproteins.
Polyclonal stimulation of resting B lymphocytes by antigen-specific T lymphocytes.
Authors: DeFranco A L , Ashwell J D , Schwartz R H , Paul W E
Published: June 2004

Resting B lymphocytes are activated, proliferate, and differentiate into antibody-secreting cells when cultured with long-term lines of major histocompatibility complex (MHC)-restricted, antigen-specific T cell in the presence of the antigen for which the T cells are specific. Under optimal conditions, essentially all B cells are activated and approximately 35% enter S phase in the absence of antigens for which the B cells are specific. Activation and proliferation are observed in cells from both normal mice and mice with the xid-determined immune defect. Highly purified B cells bearing Ia molecules for which the T cells are "cospecific" can present antigen to T cells with the resulting T cell stimulation leading to the activation and proliferation of the antigen-presenting B cells. However, B cells that do not bear Ia molecules for which the T cells are cospecific are also activated and proliferate if antigen and a source of antigen-presenting B cells or macrophage-rich cells of proper histocompatibility type are present. Thus, resting B cells, both normal and "xid", can be activated by non-MHC restricted factors without receptor cross-linkage. Experiments are presented that support the concept that local production and action of such unrestricted activating factors may be responsible for the MHC-restriction of T cell-B cell interaction seen in many circumstances.
Binding specificity of serum amyloid P component for the pyruvate acetal of galactose.
Authors: Hind C R , Collins P M , Renn D , Cook R B , Caspi D , Baltz M L , Pepys M B
Published: June 2004

Serum amyloid P component (SAP) is a normal plasma protein that is of interest because of its presence in amyloid deposits, its presence in normal human glomerular basement membrane, and its stable evolutionary conservation. It has calcium-dependent ligand-binding specificity for amyloid fibrils, fibronectin (Fn), C4-binding protein (C4bp), and agarose. Although the binding to agarose, a linear galactan hydrocolloid derived from some marine algae, is unlikely per se to be related to the physiological function of SAP, it does provide a model system in which to explore the precise ligand requirements of SAP. We report here that the amount of SAP from human, mouse, and plaice (Pleuronectes platessa L.) serum able to bind to agarose from different sources reflect precisely their pyruvate content. Methylation with diazomethane of the carboxyl groups in the pyruvate moiety of agarose completely abolishes SAP binding to agarose. The pyruvate in agarose exists as the 4,6-pyruvate acetal of beta-D-galactopyranose. We have therefore synthesized this galactoside, using a novel procedure, established its structure by analysis of its nuclear magnetic resonance spectra, and shown that it completely inhibits all known calcium-dependent binding reactions of SAP. The R isomer of the cyclic acetal, methyl 4,6-O-(1-carboxyethylidene)-beta-D-galactopyranoside (MO beta DG) was effective at millimolar concentration and was more potent than its noncyclic analogue, while pyruvate, D-galactose, and methyl beta-D-galactopyranoside were without effect. The autologous protein ligands of SAP presumably, therefore express a structural determinant(s) that stereochemically resembles MO beta DG. Availability of this specific, well-characterized, low molecular weight ligand for SAP should facilitate further investigation of the function of SAP and its role in physiological and pathophysiological processes.
Streptococcal M6 protein expressed in Escherichia coli. Localization, purification, and comparison with streptococcal-derived M protein.
Authors: Fischetti V A , Jones K F , Manjula B N , Scott J R
Published: June 2004

Type 6 streptococcal M protein produced by E. coli bearing plasmid pJRS42.13 (ColiM6) accumulates in the periplasmic space of this new host. No immunoreactive M protein was found either on the surface of the organism or in the culture medium. The ColiM6 protein was purified from the periplasm and the final preparation consisted of three protein bands of apparent molecular weight 55,000, 57,000, and 59,000. These three bands were identical in migration in SDS PAGE to that of the M protein present in freshly prepared crude periplasm. The amino acid composition of the ColiM6 protein was nearly identical to that of M protein isolated from streptococci with phage lysin (LysM6). Furthermore, except for the amino terminal residue of the LysM6 molecule, the amino terminal sequence of the ColiM6 molecule was identical to those of both LysM6 and M protein released from the streptococcus by limited peptic digestion (PepM6). These results reveal that the molecule produced in the E. coli and transported into the periplasm may be the complete M protein as it exists on the streptococcus. The results also indicate that the systems that process M protein for transport through the cytoplasmic membrane are similar in the streptococcus and E. coli. The purified ColiM6 protein was able to remove opsonic antibodies from both human and rabbit serum, as well as to stimulate the production of opsonic antibodies in rabbits, indicating that the immunodeterminants on this molecule are the same as those found on streptococcal-derived M molecules.
Rat lymphoid cell lines with human T cell leukemia virus production. I. Biological and serological characterization.
Authors: Tateno M , Kondo N , Itoh T , Chubachi T , Togashi T , Yoshiki T
Published: June 2004

Cocultivation of spleen cells, lymph node cells, and thymocytes of female Wistar-King-Aptekman rats with short-term cultured male adult T cell leukemia (ATL) cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) resulted in the establishment of rat lymphoid cell lines, TARS-1, TARL-2, and TART-1. Cytogenetic analysis of the three cell lines showed a female rat karyotype with 42 chromosomes. The surface phenotypes of TARS-1 and TART-1 were those of rat T cells. TARL-2 was only positive for rat Ia and leukocyte common antigens. The cell lines continuously produced a type C retrovirus, human T cell leukemia virus (HTLV) and expressed ATL-associated antigens. TARS-1 and TART-1, but not TARL-2 were transplantable into newborn syngeneic rats and nude mice. These results strongly indicate that HTLV not only immortalizes, but also transforms rat T cells in vitro. Adult rats immunized with either TARS-1 or TARL-2 produced antibodies specific for HTLV. The biochemical analysis of the antigens that reacted with rat sera revealed that they are the two HTLV-specific polypeptides, p24 and p28.
Monoclonal antibodies against human T cell leukemia-lymphoma virus (HTLV) p24 internal core protein. Use as diagnostic probes and cellular localization of HTLV.
Authors: Palker T J , Scearce R M , Miller S E , Popovic M , Bolognesi D P , Gallo R C , Haynes B F
Published: June 2004

Four monoclonal antibodies, human T cell leukemia-lymphoma virus (HTLV) 6, 7, 8, and 9, which react with the 24,000 dalton internal core protein of HTLVI, have been developed. These monoclonal antibodies reacted with only HTLV-infected cells and not with a broad spectrum of normal, neoplastic, mitogen-stimulated, or virus-infected cells and tissues. HTLV 6, 7, 8, and 9 identified at least two different antigenic determinants on HTLV p24 that were also recognized by antibodies present in HTLV+ patient sera. Monoclonal antibodies HTLV 6, 7, 8, and 9 reacted in indirect immunofluorescence assays with HTLV p24 localized at the cell surface of 5-d cultures of HTLV-infected T cells and, as well, reacted with T cells infected with HTLVII, a new type of HTLV isolated from a patient (MO) with a T cell variant of hairy cell leukemia. Thus, HTLV 6, 7, 8, and 9 should prove to be useful diagnostic reagents in the identification of HTLV- and HTLVII-infected T cells.
Natural resistance of lethally irradiated F1 hybrid mice to parental marrow grafts is a function of H-2/Hh-restricted effectors.
Authors: Daley J P , Nakamura I
Published: June 2004

The natural resistance of F1 hybrid mice against parental bone marrow grafts is thought to be mediated by natural killer (NK)-like effector cells. However, unlike the NK cell activity against a wide range of tumors and normal cells, hybrid resistance is characterized by the immunogenetic specificity controlled by a set of unique noncodominant genes denoted as Hh. Two alternative hypotheses can account for the specificity. Thus, the specificity may reflect either the Hh restriction of effectors or the Hh gene control of mechanisms regulating non-Hh-restricted effector activity. In this study, therefore, we tested the recognition specificity of putative effectors mediating hybrid resistance in lethally irradiated H-2b/d and H-2b/k F1 hybrid mice to the engraftment of parental H-2b bone marrow. As a direct means of defining the effector specificity, rejection of parental bone marrow grafts was subjected to competitive inhibition in situ by irradiated tumor cells. Of the 16 independent lines of lymphoma and other hemopoietic tumor cells tested, the ability to inhibit hybrid resistance was the exclusive property of all tumors derived from mice homozygous for the H-2Db region, regardless of whether the tumor cells were susceptible or resistant to NK cell-mediated cytotoxicity in vitro. Four cell lines heterozygous for the H-2Db were noninhibitory, including one that is susceptible to natural killing. Pretreatment of the F1 hosts with an interferon inducer augmented the resistance with no alteration in the recognition specificity of effector cells. Therefore, natural resistance to parental H-2b bone marrow grafts was mediated by effectors restricted by the H-2Db/Hh-1b gene(s), and not by the nonrestricted NK cells detectable in conventional in vitro assays.
Antigen-specific suppression of human antibody responses by allogeneic T cells. I. Frequency and antigen specificity of allogeneic suppressor T cells and their role in major histocompatibility complex-controlled genetic restriction.
Authors: Callard R E , Winger C M , Tiernan S L
Published: June 2004

Specific antibody responses to influenza virus were obtained in vitro from human blood mononuclear cells (PBM). The addition of allogeneic T cells to responding PBM profoundly suppressed antigen-induced antibody responses, but had no effect on pokeweed mitogen (PWM)-induced polyclonal Ig formation. This raised the possibility that suppression by allogeneic T cells may result from the activation of antigen-specific T suppressor (Ts) cells rather than nonspecific allogeneic effects. The frequency of allogeneic Ts in PBM from a number of different donors, estimated in a series of limiting dilution analyses, was found to range from 0.8 X 10(-5) to 4.5 X 10(-5), which is more typical of antigen-specific than alloreactive T cells. By adding limiting numbers of allogeneic T cells to antibody-forming cultures stimulated simultaneously with two non-cross-reacting antigens (influenza A and B strain viruses A/X31 and B/HK), it was possible to demonstrate suppression of the response to one antigen, but not the other, in the same culture well. Moreover, the frequency of wells in which the response to both antigens was suppressed was not significantly different from that predicted from the calculated frequency of specific allogeneic Ts. These results show that allogeneic suppression was antigen specific, and was not due to non-specific allogeneic effects. By separating T cell preparations into Leu-3a+ (helper) and Leu-2a+ (suppressor/cytotoxic) T cell subsets, suppression was shown to be mediated by a radiosensitive Leu-2a+ T cell. The removal of Leu-2a+ cells from T cell preparations abrogated the suppressor effect and permitted T cell collaboration with B cells, across an HLA-A, -B, and -DR barrier. This result shows that in at least some combinations, suppression rather than major histocompatibility complex restriction is the reason for the failure of allogeneic T and B cells to collaborate in T cell-dependent antibody responses.
Invertebrate recognition protein cross-reacts with an immunoglobulin idiotype.
Authors: Vasta G R , Marchalonis J J , Kohler H
Published: June 2004

To estimate the minimal structural requirements for cross-reaction of idiotypic determinants, we determined the capacity of monoclonal antibodies specific for the idiotype of the phosphorylcholine (PC)-binding myeloma protein TEPC-15 for cross-reactivities with the PC-binding, acute-phase protein C-reactive protein (CRP) and the hemagglutinin from the horseshoe crab Limulus polyphemus (limulin), which binds sialic acid and PC. Certain monoclonal antibodies (MAb) to the TEPC-15 idiotype showed strong cross-reactions with CRP and limulin when tested by enzyme-linked immunoadsorbent assays. The specificity of the cross-reactivities was confirmed by testing the binding of the reactive anti-TEPC-15 MAb to both CRP and limulin in the presence of p-nitrophenylphosphorylcholine (pNPPC), N-acetylneuraminic acid, and bovine submaxillary mucin. The binding of the MAb to both CRP and limulin was strongly decreased by pNPPC, partially decreased by free PC, and not affected by N-acetylneuraminic acid or bovine submaxillary mucin. Neither CRP nor limulin showed significant overall sequence homology to vertebrate immunoglobulins. However, CRP, limulin, and TEPC-15 variable region heavy chain (VH) shared short stretches of homology (8-10 amino acids) that mapped to a stretch comprised of the second complementarity determining region and third framework region of the TEPC-15 VH. These results might reflect either evolutionary convergence forced upon molecules of diverse evolutionary histories because of steric requirements of binding the same ligand, or a conservation of primitive combining site gene segments in evolution.
Generation and decay of the immune response to a progressive fibrosarcoma. II. Failure to demonstrate postexcision immunity after the onset of T cell-mediated suppression of immunity.
Authors: Bursuker I , North R J
Published: June 2004

This study shows that surgical removal of the meth A fibrosarcoma from its semisyngeneic host fails to result in postexcision immunity to growth of a tumor implant unless the host already has acquired a mechanism of concomitant immunity to growth of an implant. Therefore, tumor excision does not cause immunity to be generated but preserves a mechanism of concomitant immunity that already exists and which otherwise would eventually undergo down-regulation under the influence of suppressor T cells. Removal of the tumor after it has grown large enough to cause the T cell-mediated suppression of concomitant immunity does not result in the reemergence of immunity. Instead, the host remains unable to generate concomitant immunity to a second tumor for a long period of time and retains, for at least 31 d, suppressor T cells able to passively transfer suppression to appropriate recipients. Like the suppressor T cells responsible for active suppression of concomitant immunity, the suppressor T cells responsible for "memory" suppression are of the Ly-1+2- phenotype. The results indicate that progressive tumor growth results in a state of immunological tolerance of tumor-specific, transplantation antigens that can persist in the apparent absence of tumor antigens.
Multiple autoantigen binding capabilities of mouse monoclonal antibodies selected for rheumatoid factor activity.
Authors: Rubin R L , Balderas R S , Tan E M , Dixon F J , Theofilopoulos A N
Published: June 2004

We report that approximately 1/4 of monoclonal rheumatoid factors produced by hybridomas derived from fusions of spleen cells from MRL/lpr/lpr mice with systemic lupus erythematosus (SLE) and arthritis exhibited multiple reactivities with other autoantigens, including dDNA , histones, and/or cytoskeletal-cytoplasmic elements. The patterns of reactivities of most of these clones differed, indicating that each had a separate B cell ancestor. Studies with eluted antibodies demonstrated that a single species of antibody molecules was responsible for the observed multiple reactivities. Inhibition experiments suggested that an antibody combining site may be large enough to accommodate dissimilar epitopes. These findings may provide further insights into the generation and extent of antibody diversity as well as the etiopathogenesis of systemic autoimmune diseases.
Selection of strongly immunogenic "tum-" variants from tumors at high frequency using 5-azacytidine.
Authors: Frost P , Liteplo R G , Donaghue T P , Kerbel R S
Published: June 2004

Highly immunogenic "tum-" (non-tumorigenic in normal syngeneic hosts) clonal variants can be selected from a variety of poorly immunogenic and highly tumorigenic mouse cell lines at very high frequencies (e.g., greater than 80%) after treatment in vitro with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We herein demonstrate that the same result can be obtained with the poorly mutagenic cytidine analogue, 5-azacytidine, a strong DNA hypomethylating agent. 5-Azacytidine and EMS were equally and comparably effective, or ineffective, in inducing tum- variants from three different highly tumorigenic mouse cell lines. Like mutagen-induced tum- variants, those obtained after 5-azacytidine treatment generated usually strong cytolytic T lymphocyte (CTL) responses in vitro, and could grow in immunosuppressed (nude mouse) hosts. However, pretreatment of the tumor cell lines with 5-azacytidine did not cause significant increases in mutations at several independent drug-resistant gene loci, whereas EMS did. It is known that treatment of cells with 5-azacytidine can induce transcriptional activation of "silent" genes through a reduction of DNA 5-methylcytosine content, a process that can also be effected by mutagenic DNA alkylating agents such as EMS and MNNG. We therefore hypothesize that an "epigenetic" mechanism (DNA hypomethylation) leading to activation and expression of genes coding for potential tumor antigens is involved in the generation at high frequency of tum- variants after "mutagen" treatment. The implications of these findings to mechanisms of tumor progression and the generation of tumor heterogeneity are discussed.