bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective Plasmodium knowlesi Mr 74,000 antigen in membranes of schizont-infected rhesus erythrocytes.
Authors: Schmidt-Ullrich R , Lightholder J , Monroe M T
Published: June 2004

The immunogenic Plasmodium knowlesi (H strain) Mr 74,000 protein in membranes of schizont-infected rhesus erythrocytes was purified on a large scale, free of other polypeptides as monitored by dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. In a limited vaccination trial, four rhesus monkeys were immunized four consecutive times with the Mr 74,000 protein and Freund's complete and incomplete adjuvants. Two monkeys were injected with adjuvant only. Upon challenge with 10(4) viable P. knowlesi schizonts of the heterologous W strain, the control monkeys developed fatal parasitemias after 7 d. In contrast, the vaccinated monkeys exhibited a delayed onset of patent parasitemias and underwent self-cure on days 14 to 16 after peak parasitemias of between 7 and 11%. The protective immunity that was induced crossed different strains of P. knowlesi. Blood smears at the time of cure demonstrated limited reinfection, as indicated by the presence of normally appearing ring and trophozoite stages. The absence of schizont stages in the peripheral blood suggested a specific interruption of the erythrocytic schizogony at that stage. Immunochemical analyses of the rhesus sera revealed antibody only against the Mr 74,000 protein after the first two immunizations. Upon repeated antigen injection, antibodies reacted with components of Mr of approximately 102,000, 140,000, and 230,000 in addition to the Mr 74,000 protein. Besides immunological cross-reactivity, relatedness between all four immune-precipitated proteins was indicated by a greater than 50% tryptic peptide homology, suggesting that the Mr 230,000 component represents a precursor protein that is cleaved within the infected erythrocyte into proteins with Mr of approximately 140,000, 102,000, and 74,000.
Three T cell hybridomas do not contain detectable heavy chain variable gene transcripts.
Authors: Kronenberg M , Kraig E , Siu G , Kapp J A , Kappler J , Marrack P , Pierce C W , Hood L
Published: June 2004

We attempted to determine whether T cells express any VH gene segments. cDNA libraries were constructed from one suppressor and two helper T cell hybridomas. Both the library construction and screening were designed to maximize detection of a wide range of VH gene segments. One screening method should detect about half of the sequenced VH genes, while the second should detect most of these genes. The probability of detecting a VH gene homologous to the probes and present at 10 copies per cell was 77% for one helper cell cDNA library, 88% for the second helper cell library, and greater than 99% for the suppressor cell library. No cDNA clones with VH gene segments were detected. From this result, we conclude that VH gene segments are not likely to encode the antigen-specific receptor in the cells we tested.
Major histocompatibility complex-controlled, antigen-presenting cell-expressed specificity of T cell antigen recognition. Identification of a site of interaction and its relationship to Ir genes.
Authors: Hansburg D , Heber-Katz E , Fairwell T , Appella E
Published: June 2004

In previous work (5,6), we have reported studies on a T lymphocyte hybridoma clone and the peritoneal exudate T cells (PETLES) from B10.A(5R) mice primed with the cytochrome c carboxyl terminal peptide (residues 81-103) of the tobacco horn worm moth (Manducca sextus). As expected, since B10.A(5R) is a low responder to pigeon fragment 81-104, it was found that the B10.A(5R) lymphocytes were unable to respond to the pigeon cytochrome c 81-104 fragment presented on syngeneic B10.A(5R) antigen-presenting cells (APC). However, these same T lymphocytes did respond to the pigeon fragment when presented on B10.A APC. Thus, some structural difference between the pigeon and moth peptides had prevented B10.A(5R) APC from effectively presenting the pigeon fragment to moth-primed B10.A(5R) lymphocytes. This structural difference was found to be the deletion of an alanine at position -103 (Ala103) from the pigeon sequence in the moth peptide. Two additional T cell specificities were created by changing residue-99. These T cell populations from the B10.A(5R) showed an identical dependence on the Ala103 deletion when B10.A and B10.A(5R) APC were compared. The relationship of APC-expressed antigen specificity and MHC-linked immune responsiveness differences was also examined. The B10.A(5R) was found to be a high responder to each of three peptides that lack Ala103 but not to the Ala103-containing analogues. B10.A mice, in contrast, respond to both types of peptides. Utilizing allogeneic antigen-presentation to B10.A PETLES by pulsed APC, it was shown that the poor response of the B10.A(5R) to the Ala103-containing peptides was, in two of three cases, not associated with any differences in T cell repertoires but due to two different APC capabilities of B10.A and B10.A(5R). The exception apparently represents a case of T cell repertoire polymorphism between B10.A and B10.A(5R) that can also affect immune responsiveness.
Helper signals in the plaque-forming cell response to protein-bound haptens.
Authors: Roehm N W , Marrack P , Kappler J W
Published: June 2004

We have demonstrated the ability of a series of murine T cell hybridomas to deliver an antigen-specific, B cell I-region-restricted helper signal in the generation of specific PFC responses to protein-bound haptens. With some hybridomas the elicitation of optimal PFC responses required the addition of nonspecific factors provided by culture supernatants of concanavalin A-stimulated (Con A SN) spleen cells. Using hapten-primed B cells depleted of both T cells and macrophages (Mphi) we have now demonstrated a requirement for three nonspecific factor preparations to substitute for spleen Con A SN in the elicitation of optimal PFC responses. The first preparation was the interleukin 1 containing culture supernatant of the Mphi tumor cell line P388D1, the second the interleukin 2 (IL-2) and B cell growth factor containing Con A SN of the T cell hybridoma FS6-14.13, and the third, the gamma interferon containing Con A SN of the T cell hybridoma FS7-20.6.18. The P388D1 and FS6-14.13 factor preparations were most effective when added at the initiation of culture, while the FS7-20.6.18 factor preparation was most effective when added at 24 h of culture. The activity of FS6-14.13 Con A SN was depleted by incubation with the IL-2-dependent T cell line HT-2. The activity of FS7-20.6.18 Con A SN was abrogated by incubation at pH 2. The results suggest that the generation of PFC responses to protein-bound haptens require at least three nonspecific factors in addition to an antigen/Ia specific helper signal.
Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1.
Authors: Sanchez-Madrid F , Simon P , Thompson S , Springer T A
Published: June 2004

Mouse Mac-1, a complement receptor-associated surface structure on macrophages, and LFA-1, a function-associated structure on lymphocytes, comprise a novel family of leukocyte differentiation antigens participating in adhesive cell interactions. Mac-1 and LFA-1 contain alpha-subunits of 170,000 and 180,000 Mr, respectively, and beta-subunits of 95,000 Mr noncovalently associated in alpha 1 beta 1 complexes. The structural relation between the alpha- and between the beta-subunits, and the location of functionally important sites on the molecules, have been probed with antibodies. Both non-cross-reactive and cross-reactive monoclonal antibodies (MAb) and antisera prepared to the purified molecules or the LFA-1 alpha-subunits were used. Reactivity with individual subunits was studied by immunoprecipitation after dissociation induced by high pH treatment, or by immunoblotting after SDS-PAGE. Cross-reactive epitopes on Mac-1 and LFA-1 were found to be present on the beta-subunits, which were immunologically identical. Non-cross-reactive epitopes that are distinctive for Mac-1 or LFA-1 were localized to the alpha-subunits. MAb to LFA-1 alpha-subunit epitopes inhibited CTL-mediated killing. Two MAb to Mac-1 alpha-subunit epitopes but not a third MAb to a spatially distinct alpha-epitope inhibited complement receptor function. Neither function was inhibited by a MAb binding to a common beta-subunit epitope. Therefore, sites of Mac-1 and LFA-1 involved in their respective adhesion-related functions, as well as distinctive structural features, have been localized to the alpha-subunits.
Stimulus specificity of the generation of leukotrienes by dog mastocytoma cells.
Authors: Goetzl E J , Phillips M J , Gold W M
Published: June 2004

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.
Idiotype-recognizing T helper cells that are not idiotype specific.
Authors: McNamara M , Kohler H
Published: June 2004

In this study T helper cells that recognize idiotypes as carriers for a hapten-specific B cell response were analyzed under limiting dilution conditions. T helper cells, induced by phosphorylcholine-hemocyanin (PC-Hy) priming, recognize trinitrophenylated TEPC-15 and MOPC-167 (TNP-T15, TNP-167) equally well. Limiting dilution analysis indicates identical frequencies of helper cells for TNP-T15 and TNP-167. Double immunization protocols using TNP-T15 and TNP-167 fail to demonstrate additive effects. Inhibition of carrier recognition in vitro using free hapten, PC, and unconjugated T15 or M167 indicates identical specificities of helper cells for T15 and M167. Collectively, these results provide strong evidence that PC-Hy priming induces only one population of idiotype-recognizing helper cells that are unable to distinguish between the T15 and the M167 idiotopes. The helper cell induction circuit was further analyzed. PC-Hy priming induces T15/167-specific helper T cells in X-linked immune defect-expressing F1 mice. This indicates that a B cell response to PC is not required to induce idiotype-recognizing T cells. Adoptive cotransfer of B cells from PC-Hy-primed mice together with normal T cells fails to induce idiotype-recognizing T cells. These results indicate the existence of a T helper1-T helper2 induction loop. In this scheme, the T helper1 cell carries T15-like receptors and the T helper2 cells, anti-T15-like receptors. Monoclonal antiidiotypic antibodies specific for T15 also induce a T15/167-recognizing T helper cell population. This finding demonstrates that idiotope-specific priming induces non-idiotype-specific T cells. Evidently, the idiotypic T cell network is based on a different selection of idiotope determinants than the selection of the B cell idiotype network.
ABPC 48 cross-reactive idiotopes in BALB/c mice. Natural and levan-induced expression.
Authors: Legrain P , Buttin G
Published: June 2004

Using monoclonal antiidiotypic antibodies, we developed a sensitive binding assay that detects molecules with one or with two idiotopes of the ABPC48 idiotype. ABPC48 cross-reactive idiotypes were thus shown to be present in substantial amounts in sera of nonimmunized mice. Levan binding sites are found on these idiotypes. During the life time of the mice, the natural anti-levan titer increases while ABPC48 idiotypic expression remains constant, suggesting different controls for these two activities. On the other hand, ABPC48 cross-reactive idiotypes participate--as minor components--in the response that follows a deliberate immunization with bacterial levan. This induction process is likely to reflect the selection of idiotopes expressed by the B cell clones preactivated in sera of nonimmunized mice rather than the activation of silent clones. We suggest that a similar situation might explain the reported emergence of ABPC48 idiotypes in animals primed with antiidiotypic antibodies and subsequently stimulated with levan.
Unbalanced X chromosome mosaicism in B cells of mice with X-linked immunodeficiency.
Authors: Nahm M H , Paslay J W , Davie J M
Published: June 2004

The immunodeficiency in CBA/N mice is reflected by abnormal development of a subset of B lymphocytes. However, it is not clear how xid, the mutant gene in CBA/N mice, affects the development of this subset. Specifically, it is not known if the xid gene influences the development of the B cell subset directly or indirectly by providing the improper developmental milieu through effects on other cells. We investigated this question using female mice heterozygous for two x chromosomal genes, xid and Pgk-1 (phosphoglycerate kinase-1). Since females are mosaic because of x chromosome inactivation, their lymphocytes can be studied for the choice of the x chromosome, using the two PGK-1 isoenzymes as the cytological marker. We find that B lymphocytes in the spleen prefer the x chromosome without xid while the remaining splenocytes and cells from other tissues do not. This suggests that xid affects B lymphocytes directly and not through their developmental milieu. Furthermore, our data suggest that the precursors for IgG1- and IgG3-producing cells may be both few and different.
Reversal of infectious mononucleosis-associated suppressor T cell activity by D-mannose.
Authors: Tosato G , Pike S E , Blaese R M
Published: June 2004

Epstein-Barr virus-induced infectious mononucleosis (IM) is associated with the activation of suppressor T lymphocytes that profoundly inhibit immunoglobulin (Ig) production in vitro. We have examined the nature of signals operating in the interaction between IM suppressor T cells and their targets, and explored the possibility that a lectin-like receptor molecule and its specific sugar might provide specificity to this interaction. When D-mannose or some of its derivatives, including alpha-methyl-D-mannoside, mannose-6-phosphate, and mannan, were added to suppressed cultures containing IM T lymphocytes and pokeweed mitogen (PWM)-stimulated normal mononuclear cells, a significant enhancement of Ig production was observed. These sugars had little or no effect on Ig production by the PWM-stimulated responder cells alone and thus the enhanced Ig production could be attributed to the reversal of suppression in the co-cultures by these sugars. This was further confirmed by the observation that the sugars were effective only if present during the first 24 h of culture, a time when IM suppressor T cells exert their principal effect. The effect of sugars on Ig production by suppressed cultures was similar to that achieved by decreasing by about fourfold the number of IM T cells in culture. The effect of the sugars is unlikely to represent a form of nonspecific toxicity, since inhibited cultures become responders in the presence of the sugar. Furthermore, toxicity restricted to the suppressor T cells is unlikely, since preincubation of the T cells with the sugars did not reduce their subsequent ability to suppress in secondary indicator cultures. In addition, there was no correlation between the effect of the sugars on T cell proliferation and their effect on T cell-mediated suppression. The reversal of suppression by sugars was dose dependent and demonstrated stereo-specificity in that L-mannose was without effect while D-mannose reversed suppression. These data indicate that D-mannose and some of its derivatives consistently reverse suppression of Ig production by IM T cells and strongly suggest a role for saccharides as critical components in the cellular receptors involved in certain physiologic immune cell interactions.
Epitopes on H-2Dd somatic cell mutants recognized by cytotoxic T cells.
Authors: Potter T A , Palladino M A , Wilson D B , Rajan T V
Published: June 2004

We have generated several cell lines that express an altered H-2Dd molecule. These cell lines, which were selected for by the failure to express the serological specificity reacting with the monoclonal antibody 34-2-12, have also undergone alterations in epitopes recognized by CTL. One of the mutants, 2.12(-4) was not killed by an allogeneic anti-Dd CTL line, CTLL-A2, even though this line was cytotoxic for the parental cell line and two other 34-2-12- mutant lines. Two of the 34-2-12- mutant lines had an identical serological profile using other monoclonal Dd antibodies, however these two mutants differed markedly in their susceptibility to cytotoxicity by CTLL-A2. In addition to the determinants recognized by allogeneic CTL we also examined the effect of the mutation on the determinants involved in restricting the anti-FITC modified-self-cytotoxic response. An anti-FITC-Dd CTL line did not react with two of the mutants and reacted only weakly with the other mutant, demonstrating not only that the Dd epitopes recognized by this cell line and the allogeneic CTL were different, but also that it is possible for a H-2 class I molecule to express epitopes recognized by allogeneic CTL but not epitopes that function as restricting elements to certain antigens. The observation that both T cell- and B cell-defined determinants were altered in these mutant cell lines is in contrast to the findings, with the mutant mouse strains which were selected for by changes in T cell-defined determinants, which show few, if any, alterations to serological specificities. Characterization of T cell-recognized epitopes expressed on serologically selected somatic cell variants may therefore prove to be most useful for the study of structure-function relationships of H-2 class I molecules.
Immune interferon induces the receptor for monomeric IgG1 on human monocytic and myeloid cells.
Authors: Perussia B , Dayton E T , Lazarus R , Fanning V , Trinchieri G
Published: June 2004

We report here that FcR for human monomeric IgG1 can be induced on cells of myeloid origin cultured in the presence of IFN gamma for 8 h. Supernatant fluids from cultures of lymphocytes infected with a variety of viruses or cocultured with cell lines have the same FcR enhancing effect as IFN gamma. We identify the factor in the supernatant fluid responsible for the induction as immune interferon. Among the different types of IFN, only the gamma type (both purified and recombinant) specifically induces the appearance of FcR for monomeric IgG1 on normal and leukemic myeloid cells but not on cells of lymphoid origin. This effect is also evident on mature PMN. We show that the specificity and the affinity of the receptor induced on HL-60 promyelocytic cells, peripheral blood monocytes, and PMN are identical to those of the receptor spontaneously present on the same cells, except for PMN, which do not spontaneously express this type of receptor. The results of inhibition experiments performed with mouse IgG of and IgG3. These results suggest that the receptor present on human monocytes different isotypes indicate that the receptor can be inhibited by murine IgG2a or immature myeloid cells, selectively inducible by IFN gamma, has a specificity similar to the FcR1 described on mouse macrophages.
Adenosine: a physiological modulator of superoxide anion generation by human neutrophils.
Authors: Cronstein B N , Kramer S B , Weissmann G , Hirschhorn R
Published: June 2004

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.
Immunoelectron microscopic study of the distribution of T cell subsets in rheumatoid synovium.
Authors: Kurosaka M , Ziff M
Published: June 2004

The perivascular mononuclear cell collections of the rheumatoid synovium were examined both at the light and electron microscopic level by an immunoperoxidase staining technique using monoclonal antibodies directed against T cell subsets. These accumulations were variable in composition and size, not only in specimens from different patients but in the same specimen. Some areas (lymphocyte-rich areas) contained mainly small lymphocytes in clusters and others (transitional areas) contained blast cells, macrophages, and plasma cells in addition to lymphocytes. The percentage of T4 staining cells correlated positively and the percentage of T8 staining cells correlated negatively with the percentage of lymphocytes in any given area. In contrast, the percentage of T4 cells correlated negatively and the percentage of T8 cells correlated positively with the percentage of macrophage-like cells in these areas. Approximately 80% of the total lymphocytes, both in the lymphocyte-rich areas and transitional areas, were T lymphocytes (OKT3 staining). In lymphocyte-rich areas, helper/inducer T lymphocytes (OKT4 staining) were predominent over suppressor/cytotoxic lymphocytes (OKT8 staining), and in such areas the mean T4:T8 ratio was 2.9. Macrophage-like cells were seen only in small numbers in this type of area. In the transitional areas, suppressor/cytotoxic lymphocytes (OKT8 staining) predominated over helper/inducer lymphocytes (OKT4 staining). In such areas the mean T4:T8 ratio was 0.8. The T8 cells in the transitional areas tended to be large in size and often had a blastic appearance, and the abundant macrophage-like cells infiltrating these areas were frequently in close contact with T8 lymphocytes. These findings indicate that the ratio of T4 to T8 lymphocytes in rheumatoid synovium varies with the type of area examined. In lymphocyte-rich collections, made up largely of quiescent small lymphocytes, T4 cells are predominant. In areas of apparent immunological reactivity, T8 cells are predominant. It is suggested that T8 cells proliferate in immunologically active areas of the synovium as a result of local stimulation of a T cell-mediated immune response.
Elevated serum levels in human pregnancy of a molecule immunochemically similar to eosinophil granule major basic protein.
Authors: Maddox D E , Butterfield J H , Ackerman S J , Coulam C B , Gleich G J
Published: June 2004

We have shown that serum levels of a molecule immunochemically similar to eosinophil granule major basic protein (MBP) are elevated in pregnant women throughout gestation. MBP levels increase during gestation and plateau at approximately 7,500 ng/ml by the 20th wk (greater than 10-fold above normal). Levels return to normal after delivery, with a T1/2 of 13.7 d. The MBP in pregnancy serum is remarkably similar to the eosinophil granule MBP in that: (a) pregnancy MBP fully inhibits the binding of radiolabeled MBP standard in a double antibody radioimmunoassay; (b) this inhibition reaction is specific for human MBP because pregnancy serum produces no inhibition of the binding of radiolabeled guinea pig MBP in the guinea pig MBP radioimmunoassay; (c) in a two-site immunoradiometric assay for MBP, slopes of dose-response curves for pregnancy serum, purified MBP, and serum from a patient with hypereosinophilic syndrome are identical, and maximal binding is comparable; (d) reduction and alkylation of pregnancy sera increases measured MBP 100-fold, as previously shown for eosinophil granule MBP in serum; and (e) the MBP in pregnancy serum demonstrates the same pattern of heat lability as has been previously reported for MBP. Four observations have raised the possibility that the eosinophil is not the source of the MBP in pregnancy serum: (a) no correlation between serum MBP level and peripheral blood eosinophil count exists in pregnant women, in contrast to previous studies of patients with eosinophilia; (b) levels of three other eosinophil-associated proteins are normal or low in pregnancy sera, whereas the serum levels of these proteins are elevated in patients with eosinophilia; (c) the slopes of dose-response curves for pregnancy sera and MBP standards differ in the double antibody radioimmunoassay; and (d) the molecule in pregnancy serum elutes from Sephadex G-50 columns at the void volume, while eosinophil granule MBP and the MBP in serum of patients with eosinophilia elute at a volume consistent with the previously established molecular weight of 9,300. These findings suggest that the MBP in pregnancy serum is derived from a source other than the eosinophil.
Studies on the syngeneic mixed lymphocyte reaction. III. Development of a monoclonal antibody with specificity for autoreactive T cells.
Authors: Hausman P B , Moody C E , Innes J B , Gibbons J J , Weksler M E
Published: June 2004

Monoclonal antibodies with specificity for autoreactive murine T cells have been developed. These antibodies inhibit proliferative response of splenic T cells activated by syngeneic spleen cells. These antibodies have no effect on the proliferative response of T cells activated by allogeneic spleen cells or PHA. The number of splenic T cells that react with these monoclonal antibodies is comparable in several normal mouse strains.
Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes.
Authors: Horwitz M A
Published: June 2004

Previous studies have shown that L. pneumophila multiplies intracellularly in human monocytes and alveolar macrophages within a membrane-bound cytoplasmic vacuole studded with ribosomes. In this paper, the formation of this novel vacuole is examined. After entry into monocytes, L. pneumophila resides in a membrane-bound vacuole. During the first hour after entry, vacuoles containing L. pneumophila are found surrounded by smooth vesicles fusing with or budding off from the vacuolar membrane and by mitochondria closely apposed to the vacuolar membrane. By 4 h, vacuoles are found less frequently surrounded by these cytoplasmic organelles, but now ribosomes and rough vesicles are found gathered about the vacuole. By 8 h, the ribosome-lined vacuole has formed. Erythromycin, at concentrations that completely inhibit the intracellular multiplication of L. pneumophila, has no effect on vacuole formation. Formalin-killed L. pneumophila also reside in a membrane-bound vacuole after entry into monocytes. In contrast to the situation with live L. pneumophila, cytoplasmic organelles are not found surrounding vacuoles containing formalin-killed L. pneumophila at any time after entry. Formalin-killed bacteria are rapidly digested, and by 4 h, few remain intact. The L. pneumophila-containing vacuole has certain features in common with other intracellular organisms that inhibit phagosome-lysosome fusion; these organisms may share a common mechanism for vacuole formation and inhibition of phagosome-lysosome fusion.
An HLA-DR5 homozygous cell line expresses two DS (I-A-like) molecules.
Authors: Karr R W , Kannapell C C , Zangara R J , Goyert S M , Silver J , Schwartz B D
Published: June 2004

Previous studies have indicated that LLA-DR homozygous cell lines express two DR molecules but only a single DS (I-A-like) molecule. This report demonstrates that an HLA-DR5 homozygous cell line expresses at least two distinct DS molecules. These two DS molecules are formed by the association of a single DS alpha chain with either of two DS beta chains. Four distinct Ia molecules have now been identified from this DR5 homozygous cell line.
V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains.
Authors: Tonnelle C , Rocca-Serra J , Moulin A , Moinier D , Fougereau M
Published: June 2004

A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level.
T cell-replacing factor for glucocorticosteroid-induced immunoglobulin production. A unique steroid-dependent cytokine.
Authors: Orson F M , Grayson J , Pike S , De Seau V , Blaese R M
Published: June 2004

Glucocorticosteroids (GCS) added to otherwise unstimulated cultures of human peripheral blood mononuclear cells (PBMC) induce the synthesis and secretion of all classes of immunoglobulin. The magnitude of this response is similar to that seen with other polyclonal B cell activators such as pokeweed mitogen (PWM), and like that of PWM, the steroid effect is dependent on both T cells and monocytes. To determine the cellular target for GCS in these cultures, separated populations of T cells and non-T cells were preincubated with steroids and then recombined. No immunoglobulin was produced in any of these preincubation experiments. As a different approach to this question, supernatants were collected from various cell populations following stimulation with PWM, concanavalin A (Con A), phytohemagglutinin (PHA), alloantigens, or GCS. These supernatants were tested for their effects on GCS-induced Ig production by B cells. Supernatants from 3-d cultures of unstimulated, as well as GCS-treated, PBMC contained a T cell-replacing factor that permitted T-depleted PBMC to produce Ig upon steroid stimulation. This supernatant factor (TRF-S) could be produced in the absence of steroid stimulation, but both the factor and GCS were necessary for the induction of Ig synthesis. Production of the TRF-S required the presence of both T cells and adherent cells in culture and was found in the highest concentrations at 3-4 d of culture. Supernatants from cultures stimulated with PWM, PHA, Con A, and alloantigens did not contain detectable TRF-S activity, and TRF-S was unable to replace helper T cells for PWM-induced Ig production. TRF-S required the presence of adherent cells in the T cell-depleted responder population for its action. Further, it was effective in inducing Ig production along with GCS in the presence of a sufficient concentration of cyclosporin A to block all T cell helper activity for primary responses of PBMC to PWM or GCS. TRF-S was inactivated by trypsin treatment, heating to 56 degrees C, freezing, lyophilization, and storage at 4 degrees C for greater than 3 wk. Its molecular weight is probably 10,000 daltons or more, since TRF-S activity is not rapidly dialyzable. These experiments indicate that GCS-induced Ig production by human B cells does not require the presence of intact T cells in the cultures and therefore the steroids are not exerting their influence directly on T suppressor or T helper cells. Furthermore, they demonstrate a previously unrecognized cytokine that induces the differentiation of human B cells to Ig production in the presence of GCS.
Entamoeba histolytica. Phagocytosis as a virulence factor.
Authors: Orozco E , Guarneros G , Martinez-Palomo A , Sánchez T
Published: June 2004

In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica.
Amino acid sequence diversity within the family of antibodies bearing the major antiarsonate cross-reactive idiotype of the A strain mouse.
Authors: Slaughter C A , Capra J D
Published: June 2004

VH region amino acid sequences are described for five A/J anti-p-azophenylarsonate (anti-Ars) hybridoma antibodies for which the VL region sequences have previously been determined, thus completing the V domain sequences of these molecules. These antibodies all belong to the family designated Ars-A which bears the major anti-arsonate cross-reactive idiotype (CRI) of the A strain mouse. However, they differ in the degree to which they express the CRI in standard competition radioimmunoassays. Although the sequences are closely related, all are different from each other. Replacements are distributed throughout the VH region and occur in positions of the chain encoded by all three gene segments, VH, DH, and JH. It is likely that somatic diversification processes play a dominant role in producing the sequence variability in each of these segments. The number of differences from the sequence encoded by the germline is smallest for antibodies that express the CRI most strongly, suggesting that somatic diversification is responsible for loss of the CRI in members of the Ars-A antibody family. There is an unusual degree of clustering of differences in both CDR2 and CDR3 and many of the substitutions are located in "hot spots" of variation. The large number of differences between the chains prohibits the unambiguous identification of positions at which alterations play a major role in reducing the expression of the CRI. However, the data suggest that the loss of the CRI is associated with a definable repertoire of somatic changes at a restricted number of highly variable sites.
Mannose-sensitive and Gal-Gal binding Escherichia coli pili from recombinant strains. Chemical, functional, and serological properties.
Authors: O'Hanley P , Lark D , Normark S , Falkow S , Schoolnik G K
Published: June 2004

Chromosomal genes encoding the MS and Gal-Gal binding properties have been cloned into separate recombinants and their respective pili characterized. Hapten inhibition of hemagglutination with synthetic carbohydrate receptor analogues and carbohydrate-adsorbed latex agglutination studies indicate that Gal-Gal and MS pili collectively exhibit the binding properties of the parent strain. MS pili migrated in SDS-PAGE with an Mr of 19 kdaltons and 17 kdaltons; the Mr of Gal-Gal pili was 17.5 kdaltons. The pili are chemically similar by amino acid composition and when the N-terminal cysteines are aligned, 8 of the 13 residues between positions 9 and 22 are homologous. Further, carboxy-terminal sequence homology was inferred from the carboxypeptidase digestion of a MS pili and the sequence of a carboxy-terminal tryptic peptide from Gal-Gal pili.
Type-specific immunogenicity of a chemically synthesized peptide fragment of type 5 streptococcal M protein.
Authors: Dale J B , Seyer J M , Beachey E H
Published: June 2004

We determined the antigenic specificity and protective immunogenicity of two chemically synthesized peptides of type 5 streptococcal M protein. The synthetic peptides, designated S-M5(1-20) and S-M5(20-40), represent the amino-terminal amino acid sequence of the native pepsin-extracted M5 molecule, which is known to contain at least one heart cross-reactive epitope. Initial studies showed that neither of the synthetic peptides was able to bind purified heart-reactive M5 antibodies. In addition, S-M5(1-20), but not S-M5(20-40), contained type-specific antigenic determinants as measured by enzyme-linked immunosorbent inhibition assays. When covalently linked to tetanus toxoid, S-M5(1-20), but not S-M5(20-40), evoked significant levels of type-specific, opsonic (and presumably protective) antibodies in rabbits without evoking heart cross-reactive antibodies.
Antibody directed to determinants of a Moloney virus derived MCF GP70 recognizes a thymic differentiation antigen.
Authors: Johnson D A , Elder J H
Published: June 2004

We prepared an antiserum to a 14-amino-acid-long synthetic peptide derived from the published nucleotide sequence of a Moloney virus-derived MCF recombinant virus. This peptide is not found in the parental Moloney virus gp70 sequence or in other ecotropic viruses so far sequenced. When several tissues of the mouse were examined by Western blot analysis for related molecules, we found that thymocytes from 2-3-mo-old mice, but not splenic T cells or bone marrow cell extracts reacted with this probe. We found that the Mr 70,000 thymic glycoprotein recognized by the anti-peptide antiserum (81-TAg) was present at equivalent levels on peanut agglutinin positive thymocytes in all mouse strains tested. These results indicate that 81-TAg is a differentiation antigen that is related to the substituted portion of an MCF retrovirus.
A tumor-associated antigen specific for human kappa myeloma cells.
Authors: Boux H A , Raison R L , Walker K Z , Hayden G E , Basten A
Published: June 2004

A monoclonal antibody (K-1-21) raised against a kappa Bence Jones protein exhibits unique binding properties to malignant plasma cells. K-1-21 is an IgG1 kappa antibody that reacts with human kappa light chains in free form, but shows no reactivity with heavy chain-associated kappa light chains. By immunofluorescence, K-1-21 binds to the surface of LICR LON/HMy2 (HMy2) kappa myeloma cells and to plasma cells from a majority (8/11) of patients with various types of kappa myeloma; it did not bind to the surface of normal cells, nor to malignant cells of non-kappa myeloma origin. Flow cytometry analysis of K-1-21 binding to HMy2 cells indicated that the surface reactivity of K-1-21 could be completely inhibited by preincubation of the antibody with purified kappa light chains, whereas no inhibition occurred after preincubation with lambda chains or intact human IgG. Thus, the epitope recognized by K-1-21 on the cell surface may be similar, if not identical, to the determinant recognized on soluble free kappa light chains, and constitutes a tumor-associated antigen with selectivity for kappa myeloma cells. K-1-21 may therefore have clinical potential in patients with kappa myeloma.
Suppressive mechanisms in alloantigen-induced T cell responses.
Authors: Beckwith M , Rich S
Published: June 2004

In this report we examined the possibility that suppression of the mixed lymphocyte response by MLR-TsF results from interference with IL-2 regulation of T cell proliferation. Two distinct processes of inhibition involving both a direct effect on IL-2-driven proliferation of responder T cells, and induction of a second-order suppressor cell (Ts2) were described. Exogenous IL-2 did not abrogate MLR-TsF-induced suppression, and activated responder cells from suppressed cultures expressed functional IL-2 receptors by IL-2 adsorption analysis. Thus, suppression is not due to lack of available IL-2 or to abnormal acquisition of receptors for IL-2 during T cell activation. In contrast, a profound MLR-TsF effect on IL-2-induced proliferation of HT2 cells as well as MLR-activated cells was observed even after presaturation of receptors with excess IL-2. These results differentiated the direct responder cell effect of MLR-TsF from its Ts2 inductive capacity, and localized the defect in responder cell proliferation to events occurring subsequent to IL-2 binding. When analyzed in terms of proposed models for hormone-receptor interactions, characteristic dose-response curves similarly predict a postreceptor defect. Examination of the Ts2 pathway of suppression revealed a late-acting inhibitory effect peaking 72 h after MLR initiation. A minor part of Ts2 activity was susceptible to exogenous IL-2, and may reflect a requirement for IL-2 during Ts2 expansion. However, the most significant component of Ts2-mediated suppression was resistant to excess IL-2, and IL-2 production was normal in Ts2-regulated cultures, thus ruling out limitation of IL-2 for responder cell use as the major mechanism of Ts2 suppression. The complete pathway of Ts2 suppression and its functional relationship to other MLR-TsF inhibitory activities is not yet fully understood. However, these results suggest that the ultimate mechanisms of alloantigen-induced suppression involve late events of the IL-2-dependent lymphokine cascade.
Transient expression of interleukin 2 receptors. Consequences for T cell growth.
Authors: Cantrell D A , Smith K A
Published: June 2004

T lymphocyte mitosis results from the interaction of interleukin 2 (IL-2) with specific receptors that appear only after appropriate immune stimulation. To assess the potential role of IL-2 receptor levels in determining the rate and magnitude of T cell proliferation, the expression of IL-2 receptors by lectin-stimulated human peripheral blood T cells was examined and correlated with T cell growth. Using biosynthetically radiolabeled IL-2 and anti-Tac, a monoclonal antibody that blocks IL-2 receptor binding, IL-2 receptors were found to accumulate slowly and asynchronously among lectin-stimulated T cells and to precede the onset of DNA synthesis. Moreover, a critical threshold of IL-2 receptor density appeared to be required before the commitment to cell cycle progression, as analyzed quantitatively by tritiated thymidine incorporation and flow cytometric analysis of cellular DNA content. Once maximal IL-2 receptor expression occurred, continued proliferation was IL-2 concentration dependent as assessed using homogenous immunoaffinity-purified IL-2. Upon removal of the activating lectin, IL-2 receptor levels progressively declined, and, in parallel, the rate of proliferation diminished. The decay of IL-2 receptors could not be attributed to IL-2-mediated down-regulation. Instead, renewed IL-2 receptor expression was dependent upon the reintroduction of the initial activating signal. Repetitive exposure to lectin resulted in a more rapid reexpression of maximal IL-2 receptor levels, which was then followed by an accelerated resumption of proliferation. Thus, the extent of T cell proliferation after immune stimulation depends upon the interplay of the IL-2 concentration available and the density of IL-2 receptors expressed, both of which are ultimately determined by antigen/lectin stimulation. The awareness of the transience and the antigen/lectin dependence of IL-2 receptor expression, together with the capacity to monitor T cell cultures for IL-2 receptor levels, should facilitate the initiation and maintenance of cloned, antigen-specific T cells in long-term culture. In addition, these findings suggest that, in vivo, the rapidity of acquisition of maximum IL-2 receptor levels by activated T cells and the duration of IL-2 receptor expression may well direct the magnitude of T cell clonal expansion and resultant immune responses.