bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential immunoregulation of granulomatous inflammation, portal hypertension, and hepatic fibrosis in murine schistosomiasis mansoni.
Authors: Olds G R , el Meneza S , Mahmoud A F , Kresina T F
Published: December 2022

The present study investigates the immunoregulation of hepatic fibrosis in experimental murine schistosomiasis. Disease parameters measured were portal pressure, hepatic granuloma area, hepatic interstitial collagen, and glycosaminoglycans. C57BL/6 mice were infected with 25 Schistosoma mansoni cercariae and administered splenocytes or serum derived from uninfected mice or chronically infect syngeneic mice at 6 and 7 wk of infection. Immunologically mediated modulation was noted in animals receiving splenocytes derived from chronically infected mice. Both a reduction in portal pressure and hepatic granuloma areas were noted. Hepatic collagen content but not glycosaminoglycan content was reduced by the administration of either lymphoid cells or serum from chronically infected mice. The isotypic profile of hepatic interstitial collagens was modulated by both the administration of serum or lymphoid cells. Augmented levels of type III collagen was noted on administration of serum derived from chronically infected mice, whereas type I collagen levels were relatively elevated on administration of splenocytes. The data indicate that immunomodulation of inflammation and hepatic fibrosis can occur in murine schistosomiasis but that fibrotic events and inflammatory processes are independently modulated.
Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes.
Authors: Palker T J , Matthews T J , Langlois A , Tanner M E , Martin M E , Scearce R M , Kim J E , Berzofsky J A , Bolognesi D P , Haynes B F
Published: December 2022

In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.
Transcriptional and post-transcriptional regulation of human MHC class II genes require the synthesis of short-lived proteins.
Authors: Maffei A , Perfetto C , Ombra N , Del Pozzo G , Guardiola J
Published: December 2022

We have studied the stability of protein factors which control the intracellular levels of human MHC class II genes in B cells. We report that human MHC class II gene transcription and mRNA stability require the synthesis of short-lived proteins which undergo rapid intracellular turnover. We found, in fact, that the amount of MHC class II mRNA produced by human B lymphoma or B lymphoblastoid cell lines abruptly decreased upon cultivation of cells in the presence of cycloheximide, a potent inhibitor of protein synthesis. Measurements of the rate of mRNA transcription in nuclei isolated from treated cells indicated that a cycloheximide-sensitive activator protein is needed to allow MHC class II mRNA transcription initiation. Likewise, comparison of mRNA turnover rate in cells treated with actinomycin D, an inhibitor of RNA synthesis, and in cycloheximide-treated cells shows that a post-transcriptional factor is required to stabilized human MHC class II mRNA by a factor of 8- to 10-fold in B cells. These results indicate that, along with the trans- and cis-acting factors required for transcriptional control, a series of signals must exist in B cells which implement a post-transcriptional level of regulation of MHC class II gene expression in B cells.
Cytotoxic T lymphocyte clones from peripheral blood and from tumor site detect intratumor heterogeneity of melanoma cells. Analysis of specificity and mechanisms of interaction.
Authors: Anichini A , Mazzocchi A , Fossati G , Parmiani G
Published: December 2022

CTL clones isolated from PBL or from tumor-infiltrating lymphocytes (TIL) of a melanoma patient (pt665) were screened for specificity on a panel including autologous tumor cells from two distinct metastases (Me665/1, Me665/2), autologous EBV-transformed B cells and 15 allogeneic cell lines of different histology. Each clone displayed a peculiar cytolytic activity ranging from lysis of most targets (PBL clone 4C4) to preferential reactivity on the two autologous metastases (TIL clone 8B3). Blocking and modulation experiments, revealed that the lysis of autologous-Tu cells by TIL clone 8B3, but not by PBL clone 4C4, could be inhibited by mAb to HLA-class I and to CD3 Ag or by CD3 complex modulation. Clone 8B3 was tested also on a panel of 25 tumor clones from Me665/2, revealing that only 4 neoplastic clones were lysed (2/4, 2/14, 2/17, and 2/51). Cold target competition experiments indicated that the uncloned autologous melanomas and one tumor clone (2/17), but no two other tumor clones (2/10, 2/15), could compete with one another for lysis by 8B3. Determination of melanin content of tumor clones from Me665/2 revealed that the four neoplastic clones recognized by 8B3 possessed much lower melanin levels than all the other 20 clones not lysed by this effector.
IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells.
Authors: Manyak C L , Norton G P , Lobe C G , Bleackley R C , Gershenfeld H K , Weissman I L , Kumar V , Sigal N H , Koo G C
Published: December 2022

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.
Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells.
Authors: Cron R Q , Gajewski T F , Sharrow S O , Fitch F W , Matis L A , Bluestone J A
Published: December 2022

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.
Developmental potential of CD4-8- thymocytes. Peripheral progeny include mature CD4-8- T cells bearing alpha beta T cell receptor.
Authors: Guidos C J , Weissman I L , Adkins B
Published: December 2022

We have used the intra-thymic transfer system to investigate the population dynamics of thymocyte and mature T cell subsets in the absence of continuing precursor input from the bone marrow. We have followed the development and life span of CD4+ and CD8+ thymocyte subsets and mature peripheral T cells from intra-thymically injected adult or fetal CD4-8- thymic precursors. Both precursor types proliferated, differentiated, and exported to peripheral lymphoid tissues alpha beta-TCR+CD4+8- and CD4-8+ progeny which formed a stable, long-lived component of the peripheral T cell pool. The production of phenotypically mature thymocytes and peripheral T cells occurred more rapidly from fetal CD4-8- precursors. CD4+8-:CD4-8+ ratios among peripheral progeny of intra-thymically-injected CD4-8- precursors were initially normal, but they steadily declined among progeny of the fetal precursors. Thus, there appear to be differences in the life span and/or proliferative capacity of mature T cells derived from embryonic vs adult progenitors. In addition to the predominant CD4+8- and CD4-8+ subsets of peripheral T cells, a minor (1 to 20%) population of Thy-1+CD3+4-8- T cells was identified among peripheral progeny of intra-thymically-injected CD4-8- thymocytes, as well as in lymph nodes of unmanipulated animals. A total of 20 to 34% of this subset expressed V beta 8+ TCR and the majority were CD5hi, Pgp-1+, and J11d-. The function and specificity of this newly identified population of thymically derived peripheral T cells remains to be investigated.
Antimitochondrial antibodies of primary biliary cirrhosis recognize dihydrolipoamide acyltransferase and inhibit enzyme function of the branched chain alpha-ketoacid dehydrogenase complex.
Authors: Fregeau D R , Davis P A , Danner D J , Ansari A , Coppel R L , Dickson E R , Gershwin M E
Published: December 2022

Antimitochondrial antibodies (AMA) recognizing the acetyltransferase (E2) of the pyruvate dehydrogenase (PDH) complex have been previously well-documented and the immunodominant epitope mapped. In this study, we demonstrate that sera from patients with primary biliary cirrhosis (PBC) react with another lipoic acid containing acyltransferase enzyme, namely the E2 of the branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Indeed, 85/120 (71%) sera from patients with PBC reacted with BCKD-E2 by immunoblotting against purified BCKD complex. In contrast, sera from patients with chronic active hepatitis or progressive sclerosing cholangitis as well as sera from healthy volunteers did not react with any component enzymes of the BCKD complex. More importantly, BCKD enzyme activity was inhibited after incubation of the BCKD complex with either PBC sera against BCKD-E2 or with affinity purified antisera to BCKD-E2. Enzyme activity was unaltered by control sera or with PBC sera that reacted with PDH-E2 but not BCKD-E2. Furthermore, immunoblots of purified mitochondria probed with PBC sera absorbed with BCKD-E2 demonstrated that BCKD-E2 and PDH-E2 are each recognized by distinct AMA populations which do not cross-react. In addition, affinity purified PBC sera against BCKD-E2 did not react with PDH-E2 nor inhibit PDH enzyme activity, thus providing further evidence that BCKD-E2 and PDH-E2 are recognized by separate AMA. These data further suggest that the BCKD-E2 epitope recognized by AMA contains, or is close to, a functional domain of this enzyme. The availability of cDNA clones encoding BCKD-E2 and PDH-E2 will allow the study of how key metabolic enzymes may be involved in the immunology and pathology of PBC.
IL-4 down-regulates IL-1 and TNF gene expression in human monocytes.
Authors: Essner R , Rhoades K , McBride W H , Morton D L , Economou J S
Published: December 2022

The effects of IL-4 on IL-1 and TNF gene expression in human peripheral monocytes (PBM) were examined. Highly purified PBM cultured for 24 to 96 h in the presence of IL-4 produce neither IL-1 nor TNF protein. RNA hybridization studies demonstrated that IL-4 does not induce transcription of IL-1 or TNF. Preincubation of PBM with IL-4 did, however, inhibit LPS-induced IL-1 and TNF production in a dose- and time-related fashion. Maximal inhibition occurred after 96 h of incubation with 200 U/ml IL-4. RNA hybridization studies demonstrated that IL-4 suppressed the expression of IL-1 alpha, IL-1 beta, and TNF mRNA. These results suggest IL-4 modulates monocyte production of TNF and IL-1 by down-regulation of gene expression. This unique property of IL-4 may be important in the regulation of the immune response.
Distinct restriction of complement- and cell-mediated lysis.
Authors: Hollander N , Shin M L , Rosse W F , Springer T A
Published: December 2022

Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.
A phospholipase A2-activating protein (PLAP) stimulates human neutrophil aggregation and release of lysosomal enzymes, superoxide, and eicosanoids.
Authors: Bomalaski J S , Baker D G , Brophy L , Resurreccion N V , Spilberg I , Muniain M , Clark M A
Published: December 2022

We have recently isolated a human phospholipase A2-activating protein (PLAP) that shares antigenic and biochemical similarities with melittin, a well characterized bee venom phospholipase-stimulatory peptide. To explore the potential mechanisms of action of PLAP that extend beyond its effects on eicosanoid synthesis, we examined its effects on the release of human neutrophil lysosomal enzymes and superoxide, and on RBC hemolysis. These results were compared to the effects of melittin, which has been reported to induce enzyme release and hemolysis. We also examined the effects of PLAP on neutrophil aggregation and chemotaxis. PLAP induced neutrophils to release beta-glucuronidase and metalloproteinase enzyme activities as well as produce superoxide ion in both a dose- and time-dependent manner. Eicosanoid synthesis inhibitors did not abrogate these responses. PLAP induced release of arachidonic acid metabolites, but this response could be abrogated by eicosanoid synthesis inhibitors. PLAP also induced neutrophil aggregation and chemokinesis, but not chemotaxis. Concentrations of PLAP that induced these responses did not induce cellular toxicity as determined by light and electron microscopy, lactic dehydrogenase release, trypan blue dye exclusion, and RBC hemolysis. In contrast, prolonged incubation with higher concentrations of PLAP induced cell death that was similar to that observed with melittin. These findings suggest that the mechanisms of action of PLAP extend beyond the eicosanoid synthetic pathway, and that disordered regulation of PLAP may be responsible, at least in part, for chronic immune and inflammatory states.
Human T cell recognition of polymorphic epitopes from malaria circumsporozoite protein.
Authors: de Groot A S , Johnson A H , Maloy W L , Quakyi I A , Riley E M , Menon A , Banks S M , Berzofsky J A , Good M F
Published: December 2022

Lymphocytes obtained from forty individuals living in a malaria endemic area of West Africa were tested for in vitro proliferative responses to peptides representing variant regions of the immunodominant T cell domain of the circumsporozoite protein (amino acids 326 to 345, referred to as Th2R, and 361 to 380, referred to as Th3R) from three distinct strains of Plasmodium falciparum. A total of 83% of the individuals responded to at least one of the six peptides tested, confirming that these epitopes are immunodominant. A much greater number of individuals than expected by chance (32% of the responders to Th2R and 27% of the responders to Th3R) reacted to all three of the variant peptides for that epitope, indicating interdependency of the T cell responses, suggestive of cross-reactivity. Nevertheless, some subjects' T cells were clearly able to distinguish each variant peptide from the others. Using EBV transformed B cells, lymphocytes from 10 of the individuals were HLA typed. In this small group, HLA DRw13 was associated with a positive response to any of the peptides, whereas there was a negative association between DQw3 and response to any of the peptides. These results, although limited by the small sample size, suggest that recognition of T epitopes may be Ir gene linked. Our findings suggest that it may be possible to broaden the immunogenicity of an anti-sporozoite malaria vaccine.
Adenovirus persistence in man. Defective E1A gene product targeting of infected cells for elimination by natural killer cells.
Authors: Routes J M , Cook J L
Published: December 2022

Human adenovirus types 2 and 5 (Ad2/5) cause persistent infections in man. Ad2/5 infection of rodent cells induces increased susceptibility to NK lymphocyte-mediated lysis that is dependent on target cell expression of Ad2/5 E1A gene products. In contrast to infected rodent cells, Ad2/5 infection of human fibroblasts and epithelial cells does not result in increased susceptibility to either human or rodent NK cell-mediated killing, despite high levels of E1A protein expression. This functional inactivity of E1A gene products in Ad-infected human cells may contribute to adenoviral persistence by rendering the NK cell response to Ad-infected cells ineffective.
The HLA-DRw8 lineage was generated by a deletion in the DR B region followed by first domain diversification.
Authors: Gorski J
Published: December 2022

Sequences were generated for the first, second, and 3'UT regions of DRw8 beta-chain genes from two cell lines differing in their T cell determined allospecificities. Both have second domain sequences homologous to the B1 locus of the DRw52 family (DR3, DR5, and DRw6) and not the B3 locus. However, the 3'UT sequence is homologous to the 3'UT region of the B3 locus of the DRw52 family, and not the B1 locus. The first domain sequences are B1-like as opposed to B3-like and show polymorphism in the region encoding the putative alpha-helical region of the DR beta-chain. The easiest interpretation is that the DRw8 haplotypes constitute a sublineage within the DRw52 group and that this lineage has arisen by a small chromosomal deletion of the region between the B1 locus and the B3 locus. This deletion included the 3'UT region of the B1 locus, the B2 pseudogene, and the 5' end of the B3 locus including the exons encoding the first and second domains. After the deletion, two changes in the first domain arose by a mutational mechanism, possibly gene conversion. One of these changes parallels one seen in the DRw11 lineage.
MHC class II transcription in different mouse cell types. Differential requirement for protein synthesis between B cells and macrophages.
Authors: Woodward J G , Omer K W , Stuart P M
Published: December 2022

Although the MHC class II genes are known to be regulated transcriptionally, the relative rates of transcription of the four classical class II genes in different cell types have not been investigated. Using nuclear transcriptional analysis, we have investigated the transcriptional rates of the class II genes in the macrophage cell line WEHI-3, normal bone marrow-derived macrophages, L-929 cells, and two different B cell lymphoma lines. Kinetic analysis of class II transcription in IFN-gamma-treated WEHI-3 cells revealed a 4-h delay, followed by a rapid increase in transcription over the next 20 h. A significant basal level of class II transcription, apparent in bone marrow derived macrophages, was also further enhanced by IFN-gamma treatment. None of the class II genes were transcribed in L cells, whereas all class II genes were transcribed constitutively in the B cell lines. In both B cell lines and macrophages, the four class II genes were found to be transcribed at different rates from one another, but the only gene showing a consistent pattern in multiple experiments was A-alpha, always showing the highest rate. We also investigated the effect of protein synthesis inhibition on class II transcription. Cycloheximide treatment of WEHI-3 cells did not inhibit IFN-gamma-induced transcription of the class II genes within 8 h, suggesting that IFN-gamma acts on pre-existing trans-acting factors, rather than inducing their synthesis. In contrast, treatment of B cells with cycloheximide for 8 h significantly reduced class II transcription, suggesting that, in B cells, continuous synthesis of a labile trans-acting factor is required for constitutive expression. These data support the notion that class II expression in B cells is mediated by trans-acting factors distinct from those found in macrophages.
Highly restricted tumor-specific antigen of radiation leukemia virus-induced murine leukemias.
Authors: Meseguer A , Obata Y , Stockert E , DeLeo A B
Published: December 2022

A tumor-specific Ag expressed by the radiation leukemia virus-induced BALB/c leukemia BALBRVC has been serologically and biochemically identified with mAb of rat origin--RH221 and RH16. These mAb recognize two distinct epitopes on a Mr 85,000 glycoprotein. Expression of the RH221/16 Ag was also detected on two radiation leukemia virus-induced C57BL/6 leukemias, B6RV1 and B6RV2. The Ag was not detected on 66 other leukemias, 16 sarcomas, or 19 normal tissues tested. The results of sequential immunoprecipitation and partial peptide analyses of the RH221/16 Ag indicate that this tumor-specific Ag is immunologically and structurally distinct from murine leukemia virus-related determinants expressed on the three RH221/16+ leukemias.
Requirement of leukocytic cell adhesion molecules (CD11a-c/CD18) in the enhanced NK lysis of iC3b-opsonized targets.
Authors: Ramos O F , Patarroyo M , Yefenof E , Klein E
Published: December 2022

Treatment of Raji or Daudi cells with human serum under conditions which allow the alternative pathway of C activation results in their C3-opsonization and enhanced sensitivity to NK-mediated lysis. The effector lymphocytes have low buoyant density, carry CD16 and HNK1 markers as well as the CD11a-c/CD18 leukocytic cell adhesion molecules. One of these molecules, made up of CD11b-CD18 (alpha- and beta-chains), is also the receptor for iC3b. We studied the role of the cell adhesion molecules in the NK effect on targets with and without C3-fragments. We focused on the E/T interaction of opsonized cells in the presence of anti CD18 mAb. mAb directed to the CD11a molecule caused 0 to 30% inhibition of the lysis of both non-opsonized and opsonized cells whereas the mAb antibody directed to the CD11c molecule had no effect. Reagents reactive with the iC3b binding site of CD11b (alpha-chain of the CR3) molecule did not alter the lysis of non-opsonized targets whereas they abrogated the C3-mediated increment of the Nk effect on opsonized cells. Two mAb preparations, 60.3 and IB4, directed to the CD18 chain shared by the three cell adhesion molecules abrogated in a dose-dependent way the lysis of both non-opsonized and opsonized targets. The 60.3 mAb inhibited the iC3b binding site of CR3 (despite its localization on the alpha-chain) and in accordance it inhibited the binding of lymphocytes to the opsonized target also. The IB4 did not affect this site and in accordance it inhibited only partially the binding of effectors to the C3 fragment carrying Raji, nevertheless it inhibited their lysis. This result indicates that the iC3b-CR3 bridge is insufficient for triggering the lysis in absence of the contact through the adhesion molecules.
Anti-CD45 inhibition of human B cell proliferation depends on the nature of activation signals and the state of B cell activation. A study with anti-IgM and anti-CDw40 antibodies.
Authors: Gruber M F , Bjorndahl J M , Nakamura S , Fu S M
Published: December 2022

The proliferation of human peripheral and tonsillar B cells stimulated with the anti-CDw40 mAb 626.1 and/or anti-IgM antibody (Ab) in the presence of anti-CD45 mAb A.1.1 was investigated. The anti-CD45 mAb suppressed the anti-CDw40-stimulated proliferation of peripheral blood B cells but had no effect on the proliferation of unfractionated tonsillar B cells stimulated similarly. When tonsillar B cells were separated according to their sizes, the anti-CDw40-induced proliferation of small tonsillar B cells was inhibited by the anti-CD45 mAb, whereas large tonsillar B cells were resistant. In contrast, anti-IgM-induced proliferation of human B cells was always significantly inhibited by the anti-CD45 mAb regardless of cell size and tissue origin. The anti-CD45 mAb also inhibited the anti-IgM-induced initial rise in intracellular [Ca2+] and the G0-G1 cell cycle transition of small tonsillar B cells. However, co-stimulation with anti-IgM/anti-CDw40 Ab resulted in the resistance to the anti-CD45 inhibitory effect on proliferation of peripheral blood B cells and the majority of tonsillar B cells. In contrast, B cell proliferation co-stimulated with anti-IgM Ab/and B cell growth factors were always suppressed by the anti-CD45 mAb. These results demonstrate that certain activational signal mechanisms utilized by anti-CDw40/anti-IgM Ab and anti-IgM Ab/B cell growth factors are different in that B cells stimulated with these agents differ in their sensitivity to the anti-CD45 mAb. Moreover, both the activational state of human B cells and the nature of activation signals given determine their response to the inhibitory signals delivered by the anti-CD45 mAb.
Monoclonal antibodies to murine CD3 epsilon define distinct epitopes, one of which may interact with CD4 during T cell activation.
Authors: Portoles P , Rojo J , Golby A , Bonneville M , Gromkowski S , Greenbaum L , Janeway C A , Murphy D B , Bottomly K
Published: December 2022

The TCR is comprised of two variable chains that confer specificity, called alpha:beta or gamma:delta, physically associated with five different molecules that comprise the complex known as CD3. Antibodies to this complex are very useful, as they react with all T lymphocytes. A rat mAb to mouse CD3 has been prepared. It reacts with 100% of T cells in all mouse strains tested but with no other cell type. It binds to the CD3 epsilon chain. This antibody activates cloned T cell lines and normal T cells, provided suitable accessory cells and signals are present. This antibody detects a determinant similar to but not identical with those detected by two previously reported hamster anti-CD3 epsilon antibodies. This antibody fixes C efficiently, and it is thus useful for depletion of T cells from bulk populations. Activation of T cells by one of the three different anti-CD3 epsilon antibodies was inhibited by the Fab fragment of anti-CD4, similar to the effects of anti-CD4 Fab on two previously reported anti-TCR V region antibodies that bind a CD3 epsilon-associated epitope. This further defines a site involving TCR V regions and CD3 epsilon with which CD4 appears to associate during T cell activation.
Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides.
Authors: Rosloniec E F , Gay D , Freed J H
Published: December 2022

Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.
Cyclosporin A is an adjuvant in murine IgE antibody responses.
Authors: Chen S S , Stanescu G , Magalski A E , Qian Y Y
Published: December 2022

Cyclosporin A (CsA) is an undecapeptide fungal metabolite and is generally regarded as a new generation of immunosuppressive drugs. We uncovered a novel immunomodulatory property of CsA as a potent immunologic stimulator in the murine IgE antibody system. The enhancement of IgE responses was observed in mice receiving as few as three daily i.m. injections before Ag priming. Our studies demonstrate the three points listed below. First, CsA potentiates murine IgE responses regardless of Ag specificities in inbred mice. A hierarchy of immunopotentiation by CsA follows the order of low, intermediate, and high IgE responder mice. Second, CsA, when administered along with Ag, exerts a thorough and long lasting impact on the Ag-specific IgE antibody response, and leads to an Ag-specific breakthrough of IgE antibody synthesis in mice rendered tolerant in the IgE antibody system by soluble Ag pretreatment or neonatal IgE treatment. Third, IgE enhancer cells become sensitive to a low dose of irradiation. Two enhancer cellular components are identified, those of the Th cells and B cells, which appear to favor the induction of IgE responses. Understanding the cellular basis of the immunopotentiating effect of CsA will provide further insight into the murine IgE antibody system.
Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome.
Authors: Kipps T J , Tomhave E , Chen P P , Fox R I
Published: December 2022

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.
Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase.
Authors: Chedid M , Shirakawa F , Naylor P , Mizel S B
Published: December 2022

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.
Regulation of the growth rate of mouse fibroblasts by IL-3-activated mouse bone marrow-derived mast cells.
Authors: Dayton E T , Caulfield J P , Hein A , Austen K F , Stevens R L
Published: December 2022

When mouse bone marrow-derived mast cells (BMMC) are cocultured with a confluent layer of mouse 3T3 fibroblasts in the presence of WEHI-3-conditioned medium, the mast cells undergo a phenotypic change toward that of a connective tissue mast cell, and the fibroblasts increase their synthesis of globopentaosylceramide. We now demonstrate that fibroblasts lose their contact inhibition and multiply such that by the 2nd and the 4th wk of coculture there are, respectively, approximately four-fold and six-fold more fibroblasts than in the cultures that are not exposed to BMMC. This in vitro increase in the number of fibroblasts is dependent on the number of mast cells (over the range of 6 x 10(4) to 1 x 10(6) BMMC/culture) initially seeded with the fibroblasts and on the concentration of WEHI-3-conditioned medium present during the coculture. That the fibroblasts also multiply in BMMC/fibroblast cocultures exposed to synthetic IL-3 or to purified IL-3 indicates that IL-3 is a component in WEHI-3-conditioned medium that induces mast cells to produce the fibroblast growth factor. The number of fibroblasts does not increase if fibroblasts are exposed to lysates of BMMC, or to BMMC-derived conditioned medium, or if the two cell types are separated from one another during the coculture with a 3-microns filter or a 0.4-microns filter. Thus, IL-3-activated BMMC must be in proximity to fibroblasts to induce them to multiply. Because of their increased numbers per culture dish, total fibroblasts that were cocultured with mast cells synthesized approximately two-fold more 35S-labeled proteoglycans, incorporated approximately 3-fold more [3H] proline into collagenase-sensitive proteins, and had substantially more alpha 2(I) collagen mRNA than fibroblasts that were maintained in the absence of mast cells. These is vitro studies reveal a sequence by which IL-3-activated mast cells may play a role in the induction of fibrosis.
Binding of model immune complexes to erythrocyte CR1 facilitates immune complex uptake by U937 cells.
Authors: Emlen W , Burdick G , Carl V , Lachmann P J
Published: December 2022

The E C3b/C4b receptor (CR1) has been shown to rapidly bind large complement-fixing immune complexes (IC) both in vivo and in vitro. It has been proposed that E (RBC) CR1 act as a shuttle mechanism, binding circulating IC and transporting them to tissue macrophages, thereby preventing their deposition in target tissues. In this study we have established an in vitro model system with which to study the transfer of model IC from CR1 on the RBC surface to phagocytic cells. Aggregated IgG (AHG) was opsonized with C3b, bound to RBC CR1, and the binding of these RBC-bound IC by a human monocyte cell line (U937 cells) was examined. U937 binding of AHG from the RBC surface was complete within 2 min, whereas binding of the same AHG from solution required 30 to 60 min. Despite the difference in kinetics of binding, the total amount of IC bound by U937 cells at equilibrium was the same for RBC-bound AHG and for AHG in solution. The transfer of AHG from the RBC to the U937 cell did not require exogenous factor I and was not accompanied by binding of RBC to U937 cells or by erythrophagocytosis. Our data lend support to the hypothesis that binding of IC to RBC CR1 may facilitate the clearance of IC from the circulation by enhancing their uptake by phagocytic cells.
Depression of polymorphonuclear leukocyte functions by purified influenza virus hemagglutinin and sialic acid-binding lectins.
Authors: Cassidy L F , Lyles D S , Abramson J S
Published: December 2022

Infection of polymorphonuclear leukocytes (PMNL) with influenza virus causes depression of PMNL metabolic and bactericidal activities. The studies reported here were undertaken to determine whether the hemagglutinin (HA) glycoprotein of influenza virus mediates this depression. PMNL were incubated with purified HA and the oxidative responses to exogenous stimuli were measured. The results indicate that HA, in either liposomes or protein aggregates referred to as rosettes, depressed PMNL oxidative responses. Depression was observed within 2 min of initial interaction of HA with PMNL and lasted more than 2 h. The membrane fusion activity of HA requires proteolytic cleavage of the HA, whereas the receptor binding activity does not. There was no difference in the ability of virions with cleaved or uncleaved HA to depress PMNL responses suggesting that the fusion event is not required for PMNL dysfunction. Inasmuch as the HA glycoprotein binds to sialic acid-containing receptors on the surface of the PMNL, we tested whether other sialic acid-specific binding proteins can mediate the reduction of PMNL responses. Sialic acid-specific lectins from Limulus polyphemus or Limax flavus were incubated with PMNL before measuring their responses to secondary stimulus. Depression was observed upon incubation with the lectins similar to that seen upon incubation with the HA or influenza virus. These results suggest that attachment of influenza virus to sialic acid-containing receptors is responsible at least in part, for suppressing PMNL oxidative responses.
IgE receptor-mediated hydrolysis of phosphoinositides by cytoplasts from rat basophilic leukemia cells.
Authors: Dreskin S C , Pribluda V S , Metzger H
Published: December 2022

Cytoplasts (plasma membrane sacs containing cytoplasm, endoplasmic reticulum, and few organelles) were prepared from rat basophilic leukemia cells by treatment with cytochalasin B and centrifugation at 33 degrees C through stepwise gradients of Ficoll. To compare the relative ability of cytoplasts and cells to generate second-messengers (inositol phosphates, Ca2+) in response to stimulation of the high affinity receptor for IgE, we normalized our results per recovered receptor by using the tightly bound IgE as a marker. This marker correlated well with other estimates of plasma membrane recovery. Furthermore, data normalized on this basis correlated well with data expressed as percentage of phosphoinositides hydrolyzed. The purest fraction of cytoplasts (containing about 6% of the receptors) was satisfactorily devoid of organelles and, at early times, generated about 50% as much inositol phosphates per receptor as did the intact, untreated cells. This response of the cytoplasts, like that of the cells, was totally dependent upon aggregation of the receptors. The response by the cytoplasts (in the 5-min time frame which we examined), unlike that of the cells, was not enhanced by the presence of extracellular Ca2+. Furthermore, unlike the cells, the cytoplasts failed to raise their intracellular free Ca2+ levels after addition of polyvalent Ag. This result suggests that aggregation of the receptors may be insufficient, by itself, to open the normal Ca2+ channels.
Variations in protein expression related to human eosinophil heterogeneity.
Authors: Gruart V , Balloul J M , Prin L , Tomassini M , Loiseau S , Capron A , Capron M
Published: December 2022

In hypereosinophilic patients, eosinophil heterogeneity has been assessed mainly according to morphologic and biologic criteria. In order to investigate the molecular basis of such heterogeneity, biochemical analysis was performed on various eosinophil subpopulations fractionated on metrizamide gradients. Whole cell extracts from purified eosinophils disrupted with a nonionic (NP-40) detergent were successively analyzed by SDS-PAGE and two-dimensional electrophoresis (isoelectric focusing or nonequilibrium pH gradient electrophoresis in the first dimension). Hypodense eosinophils that sediment in the lightest density gradients (18 to 22% metrizamide solution) differed from other purified eosinophils (intermediate and normodense eosinophils respectively collected in 22 to 23% and 23 to 25% metrizamide solutions). Comparative analysis of protein patterns on both monodimensional and bidimensional electrophoresis showed that a basic protein of Mr 51 kDa, present on normodense or intermediate eosinophils, was poorly detected in the case of hypodense eosinophils. In contrast, two other proteins with apparent Mr of about 23 kDa and 41 kDa were exclusively or predominantly identified in these latter cell fractions. Immunochemical analysis with polyclonal antibodies against eosinophil basic proteins and enzymatic assays revealed that the 51-kDa polypeptide could be related to an eosinophil peroxidase-like molecule. In addition, the two proteins detected only in hypodense eosinophils might be related to proteins newly synthesized by in vivo activated eosinophils. Our results suggest that variations in protein expression might represent a good marker of in vivo activation.
Functional role of human IgG subclasses in eosinophil-mediated killing of schistosomula of Schistosoma mansoni.
Authors: Khalife J , Dunne D W , Richardson B A , Mazza G , Thorne K J , Capron A , Butterworth A E
Published: December 2022

Although IgG antibodies and eosinophils have been shown to kill schistosomula of Schistosoma mansoni in vitro, very little data exist that describe the role of each IgG antibody isotype in this event. This study was designed to test the role of each IgG subclass in the eosinophil-dependent killing reaction. IgG antibodies purified by protein G or protein A affinity chromatography demonstrated a killing effect only in the presence of eosinophils activated in vivo or normal eosinophils activated in vitro by eosinophil activating factor. Purification of each IgG isotype allowed confirmation of these results and demonstrated that the killing effect was associated with IgG1 and IgG3 antibodies. IgG2 antibodies expressed a dual function: 1) an effector function with activated eosinophils and 2) a blocking function with normal eosinophils. IgG4 antibodies, whatever the source of eosinophils, blocked the killing mediated by IgG effector antibodies. These findings are discussed in relation to immunity and susceptibility to reinfection in human schistosomiasis.