bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-gamma regulates the expression of the adhesion molecule ELAM-1 and IL-6 production by human endothelial cells in vitro.
Authors: Leeuwenberg J F , von Asmuth E J , Jeunhomme T M , Buurman W A
Published: December 2022

In this study two new in vitro effects of IFN-gamma on human umbilical vein endothelial (HUVE) cells were described. First, it was shown that the expression of the adhesion molecule ELAM-1 on activated HUVE cells can be modulated by IFN-gamma. ELAM-1 is normally not expressed by HUVE cells, but its expression can rapidly be induced by TNF, IL-1, or LPS. Maximal expression is reached after 4 to 6 h of activation, and after 24 h the expression disappeared. Whereas IFN-gamma per se did not induce expression of ELAM-1, it enhanced and prolonged the expression of ELAM-1. This enhancement occurred when IFN-gamma was added before activation as well as when added simultaneously with activation. When IFN-gamma was added 6 or 9 h after the activation, the normally ongoing reduction of expression was not only retarded, but the expression increased for at least 3 h. Moreover, IFN-gamma abrogated the refractory period for restimulation. Neither IFN-beta nor IL-6 had any effect on the expression of ELAM-1. The second effect of IFN-gamma on HUVE cells is the capacity to enhance the IL-6 production by these cells. Prestimulation as well as coincubation of IFN-gamma with TNF, IL-1, or LPS resulted in a strongly augmented production of IL-6. The effects of IFN-gamma may in vivo play a role in the regulation of an inflammatory reaction, because ELAM-1 is an adhesion molecule for neutrophils, and IL-6 has an enhancing effect on the cytotoxicity of neutrophils.
Thymic B cells from myasthenia gravis patients are activated B cells. Phenotypic and functional analysis.
Authors: Leprince C , Cohen-Kaminsky S , Berrih-Aknin S , Vernet-Der Garabedian B , Treton D , Galanaud P , Richard Y
Published: December 2022

Thymic cell populations from 12 patients displaying myasthenia gravis were submitted to a phenotypic and functional study. Immunofluorescence analysis of thymic sections revealed the presence in germinal centers of B lymphocytes expressing the B cell markers--CD19, CD21, IgD, or IgM. After T cell and macrophage depletion of thymic single cell suspensions, B cell-enriched populations were isolated. Enriched B cells expressed at variable levels activation markers such as CD71, 4F2, CD23, and B8.7, indicating that a marked proportion of them are activated. Moreover, addition of B cell growth factor 12kDa and to a lesser extent of rIL-2 induced a spontaneous proliferation of these B cell populations. These functional and phenotypic signs of activation may reveal the first steps of an autoimmune response against acetylcholine receptor as enriched B cell populations have the capacity to spontaneously secrete anti-acetylcholine receptor antibody.
Adverse immune reactions to gold. I. Chronic treatment with an Au(I) drug sensitizes mouse T cells not to Au(I), but to Au(III) and induces autoantibody formation.
Authors: Schuhmann D , Kubicka-Muranyi M , Mirtschewa J , Günther J , Kind P , Gleichmann E
Published: December 2022

Upon weekly i.m. injections of disodium gold thiomalate (Na2AuTM) 100% of A.SW mice produced IgG autoantibodies to antinuclear Ag and nucleolar Ag, respectively; about 70% of C57BL/6 mice produced IgG antinuclear Ag, whereas DBA/2 mice were resistant. Moreover, C57BL/6 mice, but not DBA/2 mice, showed increased mesangial deposits of IgG. These alterations were due not to disodium thiomalate, but to the gold ion of Na2AuTM. An assumed T cell reactivity of susceptible mouse strains to Na2AuTM was tested by means of the direct popliteal lymph node (PLN) assay. However, no distinct PLN reaction to Na2AuTM was detectable. Likewise, AuCl did not induce a PLN reaction. Both Na2AuTM and AuCl contain gold in the Au(I) state. The poor PLN responses to Au(I) contrasted with the strong PLN responses to Au(III) compounds. PLN reactions to Au(III) were dose dependent, T cell dependent, and specific. When Au(III) was reduced to Au(I) by addition of Na2TM or methionine before testing in the PLN assay its sensitizing capacity was significantly decreased. Thus, the oxidation state of gold, i.e., Au(III) vs Au(I), plays a major role for its sensitizing capacity. Therefore, we propose that the Au(I) of Na2AuTM is oxidized to Au(III) before T cells are sensitized and adverse immunologic reactions develop. Results obtained with the adoptive transfer PLN assay indicated that, indeed, repeated i.m. injections of Na2AuTM sensitized A.SW and C57BL/6 splenic T cells to Au(III).
TAPA-1, the target of an antiproliferative antibody, is associated on the cell surface with the Leu-13 antigen.
Authors: Takahashi S , Doss C , Levy S , Levy R
Published: December 2022

A murine mAb, 5A6 (IgG1), has been isolated by immunization with a human B lymphoma cell line and screening for growth inhibition. The antibody immunoprecipitated a single chain protein of 26 kDa from cell lysates made with Triton X-100 but additional proteins were precipitated when cell lysates were made with the milder detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio)-1-propane sulfate). We have identified one of these coprecipitated molecules as the 16-kDa Leu-13 Ag. 5A6 and anti-Leu-13 showed similar, although not identical, reactivity, growth inhibition and temperature-dependent aggregation effects among hematolymphoid cell lines. The aggregation induced by 5A6 and anti-Leu-13 was not dependent on LFA-1 (lymphocyte function-associated Ag-1). The cell-surface expression of both TAPA-1 (target of an antiproliferative antibody-1) and Leu-13 could be down-modulated by binding to their respective antibodies and they could be reciprocally comodulated. These results suggest that TAPA-1 and Leu-13 form a complex on the cell surface and play a role in growth control through a common pathway.
Evidence that increasing the cellular content of eicosapentaenoic acid does not reduce the biosynthesis of platelet-activating factor.
Authors: Triggiani M , Connell T R , Chilton F H
Published: December 2022

Our study has examined platelet-activating factor (PAF) biosynthesis in neutrophils from individuals on a fish oil-enriched diet and in mast cells enriched with eicosapentaenoic acid (EPA) in vitro. Neutrophils isolated from males who were fed fish oil supplement (EPA; 2.8 g/day) for 5 wk contained large quantities of eicosapentaenoate in phosphatidylcholine (PC) and phosphatidylethanolamine and less in phosphatidylinositol. The ratio arachidonate/eicosapentaenoate in PC and phosphatidylethanolamine decreased from greater than 10 before the enriched diet to approximately 3 after the diet. The putative precursor of PAF, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC) contained the bulk of eicosapentaenoate in PC subclasses with smaller quantities found in 1-acyl and 1-alk-1'-enyl linked species. Ionophore A23187-stimulated neutrophils produced similar quantities of PAF before and after enriched diet. Neutrophils during normal diet acylated 1-O-alkyl-2-lyso-GPC only with arachidonate whereas neutrophils from individuals on enriched diet transferred both arachidonate and eicosapentaenoate into exogenously-provided 1-O-alkyl-2-lyso-GPC. This allowed for the labeling of neutrophils with 1-O-[3H]-alkyl-2-arachidonoyl-GPC (before diet) as well as neutrophils with 1-O-[3H]-alkyl-2-eicosapentaenoyl-GPC and 1-O-[3H]-alkyl-2-arachidonoyl-GPC (after diet). Neutrophils after diet converted similar quantities of these labeled precursors to labeled PAF upon stimulation as those before the diet. Analysis of the nature of the long chain acyl residue remaining in the sn-2 position of 1-alkyl-2-acyl-GPC after cell stimulation indicated that arachidonate and eicosapentaenoate were both released from 1-O-alkyl-2-acyl-GPC at comparable rates. Finally, in vitro supplementation of murine mast cells (PT-18) with arachidonic acid or EPA caused a marked increase in the amount of PAF produced by the cell without having any effect on histamine release. Data from these experiments suggest that EPA is incorporated into a PAF precursor pool. However, this appears not to inhibit PAF production because phospholipase A2 can use eicosapentaenoate- as well as arachidonate-containing phospholipids in the initial step of PAF biosynthesis.
Identification of an HSV-1/HSV-2 cross-reactive T cell determinant.
Authors: Grammer S F , Sette A , Colón S , Walker L , Chesnut R
Published: December 2022

The results from a number of studies have documented that the HSV glycoprotein gD is an important target for neutralizing antibodies. In contrast, little is known about the Th cell determinants present on HSV that are required for anti HSV gD antibody production. In our study we have immunized BALB/c mice with a recombinant source of HSV-1 gD lacking the carboxyl-terminal 93 amino acids. T cell hybridomas produced from the immunized animals recognized a single antigenic peptide (amino acids 246-261) in the context of I-Ad. The determinant expressed by gD peptide 246-261 was generated and presented by both HSV-1 and HSV-2 infected APC. Fine specificity analysis using truncated synthetic gD peptides revealed that the minimal amino acids recognized by the T hybrids were identical between HSV-1 and HSV-2. In addition, the minimal peptide-I-Ad binding analysis demonstrated that the minimal peptide sequence required for the binding to I-Ad and for T cell recognition contained two prolines. Thus, this important HSV antigenic determinant would not be expected to form an amphipathic alpha-helix and could therefore be missed by algorithms currently used to predict which amino acid sequences would be antigenic based on the propensity to form helices.
Induction of immediate early response genes by macrophage colony-stimulating factor in normal human monocytes.
Authors: Mufson R A
Published: December 2022

A group of coordinately induced protooncogenes, cytoskeletal, and extracellular matrix genes have been termed immediate early response genes, and their induction has been associated with growth factor-stimulated cell proliferation. We have investigated the induction of these genes by macrophage-CSF (M-CSF) in human monocytes that do not proliferate in response to M-CSF but require the factor for optimal cell differentiation. Normal human monocytes were isolated, carefully washed, and incubated for 36 to 48 h in fetal bovine serum-containing medium. At the end of this incubation the resting cells were stimulated with M-CSF, and RNA was isolated for analysis by Northern blotting. RNA from control resting cells contained low to undetectable levels of c-jun, fibronectin receptor, and actin mRNA. Within 15 to 30 min of addition of M-CSF, however, there was a dramatic coordinate induction of these genes. The c-jun gene expression was very transient and was not detectable by 60 min after M-CSF addition. In contrast, the expression of actin and fibronectin receptor mRNA was more sustained, and the expression of these genes remained elevated at 24 to 48 h after M-CSF addition. We also observed the induction of the myelomonocytic specific tyrosine kinase hck gene simultaneously with the other immediate early response genes. The protein synthesis inhibitor cycloheximide did not block the induction of any of these genes, and in fact, super-induced the expression of c-jun and hck. Nuclear run on transcription of the c-jun, hck, and actin genes. Therefore, in normal human monocytes M-CSF induces immediate early response genes without inducing cell proliferation. These genes may then play a role in altering the physiologic status of the cells in response to CSF.
Gene deletions force nonsecretory alpha-chain disease plasma cells to produce membrane-form alpha-chain only.
Authors: Cogné M , Preud'homme J L
Published: December 2022

We studied a case of nonsecretory alpha-chain disease. The proliferating plasma cells contained a short transcript coding for a truncated membrane-form alpha 1-chain. The productive alpha-gene bore several noncontiguous deletions affecting the VHDJH and CH1 regions. Two deletions were accompanied with peculiar insertions containing duplications. The first insertion contained an acceptor splice site and was present in part in the mature transcript, thus coding for an abnormal aminoterminal peptide. Another deletion located 3' to CH3 eliminated the polyadenylation site of secreted-form alpha-mRNA. As a result, only membrane-form alpha mRNA was present in the tumoral plasma cells, thus explaining the nonsecretory phenotype of the disease. Comparison of cDNA and genomic sequences showed that the previously undescribed human alpha membrane region is encoded by a single exon, beginning with two alternate acceptor splice sites, and comprises either 65 or 71 amino acids.
IL-3 promotes production of IL-4 by splenic non-B, non-T cells in response to Fc receptor cross-linkage.
Authors: Le Gros G , Ben-Sasson S Z , Conrad D H , Clark-Lewis I , Finkelman F D , Plaut M , Paul W E
Published: December 2022

A spleen cell population that lacks CD3, CD4, CD8, Thy-1, B220, and class II major histocompatibility complex cell-surface markers (non-B, non-T cells) produces IL-4 when cultured in wells coated with IgE. Their production of IL-4 in response to plate-bound (PB)-IgE is strikingly enhanced by IL-3, and in the presence of IL-3, these cells also produce IL-4 in response to PB-IgG2a. The effect of IL-3 is not mimicked by IL-1, IL-2, IL-5, IL-6, IL-7, granulocyte-macrophage CSF (GM-CSF) or IFN-gamma. Non-B, non-T cells cultured with IL-3 for 12 h acquire the capacity to produce enhanced amounts of IL-4 in response to subsequent culture with PB-Ig even if IL-3 is omitted from the second culture. Irradiated cells also respond to IL-3 with enhanced capacity to produce IL-4 to PB-Ig, indicating that cell proliferation is not required for the effect of IL-3. The IL-3 effect can be obtained in vivo; treatment of mice with a total dose 90,000 U of synthetic IL-3 over a 3-day period results in the presence of splenic and peritoneal cavity non-B, non-T cells that produce enhanced amounts of IL-4 in response to PB-Ig. The FcR that mediates the response to PB-IgE appears to be Fc epsilon RI because cells can be sensitized with IgE anti-DNP mAb, washed, cultured for 15 h at 37 degrees C, washed again, and stimulated to produce IL-4 with 0.1 to 1 ng/ml of TNP10-OVA. IL-3 does not appear to mediate its function by increasing the number of Fc epsilon RI because it can exert its effect when cultured with non-B, non-T cells after they have been sensitized with IgE anti-DNP. However, IL-3 pretreatment does affect the signaling process in that non-B, non-T cells sensitized with IgE anti-DNP show strikingly reduced production of IL-4 to concentrations of TNP10-OVA of 100 ng/ml or more whereas cells pretreated with IL-3 show little or no diminution in IL-4 production at concentrations of TNP10-OVA up to 1 microgram/ml.
An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein.
Authors: Zhou S R , Whitaker J N
Published: December 2022

A mAb of the IgG1/kappa isotype was raised against human myelin basic protein (MBP) peptide acetyl 1-9. This mAb, termed F23, reacted with human MBP and human MBP peptides acetyl 1-9, 1-14, and 1-44, but not with MBP peptides 10-19, 80-89, or 45-89. According to the guidelines of the molecular recognition theory, a complementary peptide to human MBP peptide 1-9 was synthesized and used to raise murine mAb with anti-Id activity. Two mAb anti-Id, F25F7 and F25C8, both of the IgM/kappa isotype, were selected for further study. These anti-Id reacted with F23, the mAb for which they were selected, and also reacted with another mAb, which was of the IgG1/kappa isotype and was raised to human MBP peptide 80-89. There was no reaction with another control mAb of the IgG1/kappa isotype or murine myeloma IgG1. By immunoblotting techniques, it was demonstrated that the Id on each of the mAbs to MBP peptides was located on the kappa L chain but also could be recognized in nonreduced IgG. The cross-reactive anti-Id suppressed antibody secretion of Id-producing hybridoma cells in an Id-specific manner, and kinetic studies suggest an intracellular mechanism for the suppression. These cross-reactive Id among antibodies to different MBP peptides imply that the same V region genes of kappa L chains are involved in the selection of antibodies to an autoantigen, like MBP, and may play a role in the modulation of immune responses against MBP in certain inflammatory demyelinating diseases.
Phenotypic and functional characteristics of in vivo-activated Langerhans cells.
Authors: Aiba S , Katz S I
Published: December 2022

After short term culture (2 to 3 days), Langerhans cells (LC) exhibit increased class II MHC Ag and become more potent APC than freshly obtained LC in primary allogeneic and syngeneic T cell activation. To determine whether in vivo LC undergo changes similar to cultured LC, we examined the phenotypic and functional characteristics of LC harvested from ear skin of naive mice painted with various haptens and primary irritants. At 24 h after application of 3% trinitrochlorobenzene, LC appear larger and exhibit more intense staining in epidermal sheets using anti-I-A antibodies, and there was a two- to threefold increase in I-A and I-E expression by LC using flow microfluorimetry analysis. CD45 Ag expression was not altered. Flow microfluorimetry profiles showed the presence of two different LC populations based on fluorescence intensity, i.e., one with the same Ia density as nontreated LC and the other (representing 22 to 50% of all LC) with a markedly enhanced Ia density, (i.e., a 10-fold increase in I-A and I-E). This phenotypic change was observed only with haptens, such as trinitrochlorobenzene, dinitrofluorobenzene, oxazolone, and cinnamic aldehyde. In contrast, application of 10 to 30% sodium lauryl sulfate or vehicle controls did not induce this change. Functionally, LC obtained from hapten-painted mice induced a two- to fivefold increase in 3[H]-TdR incorporation by syngeneic or allogeneic T cells, compared to equal numbers of LC from nontreated or vehicle-treated or sodium lauryl sulfate-treated mice. These phenotypic and functional changes that occur in vivo are therefore analogous to those seen when LC are cultured for short periods of time. Thus, activated LC appear in vivo in response to the epicutaneous application of haptens and may represent an essential step in hapten-specific sensitization.
Expression of functional alpha beta T cell receptor gene rearrangements in suppressor T cell hybridomas correlates with antigen binding, but not with suppressor cell function.
Authors: Collins M , Kuchroo V K , Whitters M J , O'Hara R M , Kelleher K , Kubo R T , Dorf M E
Published: December 2022

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.
Human cytotoxic lymphocytes. V. Frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors activated by allogeneic or autologous stimulator cells.
Authors: Kabelitz D , Bender A , Schondelmaier S , da Silva Lobo M L , Janssen O
Published: December 2022

We have investigated the frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors (CLP) under limiting dilution culture conditions. E rosette separated total T cells and CD3+CD4-CD8-TCR alpha beta- double-negative (DN) T cells were cocultured with allogeneic or autologous PBMC stimulator cells, and frequencies of alloreactive and autoreactive CLP were determined after 12 to 14 days against Con A blast target cells. Freshly isolated DN cells consisting of 82.3 +/- 8.2% gamma delta+ T cells did not exert cytolytic activity against K562 or anti-TCR gamma delta mAb-producing hybridoma cells. In striking contrast to E+ cells, the vast majority of alloantigen-stimulated clonally developing DN CLP did not show specificity for stimulator-derived target cells. Thus, frequencies of alloreactive and autoreactive CLP after alloantigenic stimulation were in the range of 1/100 to 1/4800 and 1/450 to 1/5000, respectively. After coculture with autologous stimulator cells, frequencies of autoreactive and alloreactive DN CLP were 1/700 to 1/2700 and 1/1360 to 1/4500, respectively. Split culture analysis revealed that most proliferating DN colonies selected for high probability of clonality simultaneously killed both autologous and HLA-mismatched allogeneic targets. The majority of the DN cells expressed the CD3+/TCR gamma delta+ phenotype after culture, and thus were not CD2+CD3- NK cells. Taken together, our results show that 1) freshly isolated peripheral blood gamma delta+ T cells lack cytotoxic activity, and 2) most cytotoxic gamma delta+ T cells activated by autologous or allogeneic stimulator cells under limiting dilution conditions do not discriminate between autologous and allogeneic targets.
Functional epitope analysis of the human CD4 molecule. The MHC class II-dependent activation of resting T cells is inhibited by monoclonal antibodies to CD4 regardless whether or not they recognize epitopes involved in the binding of MHC class II or HIV gp120.
Authors: Merkenschlager M , Buck D , Beverley P C , Sattentau Q J
Published: December 2022

This study was designed to define regions on the human CD4 molecule important for the class II-dependent activation of resting, polyclonal CD4 T cells. With the use of mAb to known epitopes on CD4, we assayed the degree of CD4 saturation and functional effects on T cell activation over a range of antibody concentrations in parallel titration experiments. This approach allows a quantitative comparison of different reagents, regardless of parameters such as affinity for CD4. In sharp contrast to results reported for preactivated T cells and CD4 transfected T cell hybridomas, all 22 CD4 mAb tested did inhibit proliferative responses of freshly isolated CD4 T cells to MHC class II Ag. At the lowest saturating concentration of each antibody, T cell proliferation was reduced by 45 to 82%. Inhibition did not depend on antibody-induced modulation of CD4 expression. Strikingly, no correlation was found between the functional effects and the specificity of the mAb for different epitopes on CD4, such as the putative binding sites for MHC class II or HIV glycoprotein gp120.
In vivo ultraviolet-exposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressor-inducer T cells.
Authors: Baadsgaard O , Salvo B , Mannie A , Dass B , Fox D A , Cooper K D
Published: December 2022

In vivo UV exposure of human epidermis abrogates the function of CD1+DR+ Langerhans cells and induces the appearance of CD1-DR+ Ag-presenting macrophages. Epidermal cells from UV-exposed skin, in contrast to epidermal cells from normal skin, potently activate autologous CD4+ T cells, and, in particular, the CD45RA+ (2H4+) (suppressor-inducer) subset. We therefore determined whether UV-exposure in humans leads to a T cell response in which suppression dominates. Autologous blood T cells were incubated with epidermal cell suspensions from in vivo UV-irradiated skin. After activation, repurified T cells were transferred in graded numbers to autologous mononuclear cells (MNC) stimulated with PWM and the resultant IgG production analyzed by ELISA. Relative to T cells activated by unirradiated control epidermal cells, T cells activated by UV-exposed epidermal cells demonstrated enhanced capacity to suppress IgG production (n = 6; p less than or equal to 0.03). Within the T cell population, CD8+ cells stimulated by UV-exposed epidermal cells could be directly activated to suppress PWM-stimulated MNC Ig production if IL-2 was provided in the reaction mixture. The suppressive activity was also transferable with purified CD4+ T cells stimulated by UV-exposed epidermal cells (n = 10; p less than or equal to 0.01), and was radiosensitive. Suppression was decreased when PWM-stimulated MNC were depleted of CD8+ T cells before mixing with CD4+ T cells activated by UV-exposed epidermal cells, suggesting indirect induction of CD8+ Ts cells contained within the responding MNC populations. Indeed, physical depletion of CD45RA+ cells resulted in total abrogation of the suppressor function contained in the CD4+ T cells. Activation of suppressor function was critically dependent on DR+ APC contained in UV-exposed epidermis. The data suggest that UV-exposure modulates cutaneous APC activity in humans, as in mice, such that the dominant immune response is tilted toward suppression. These mechanisms in normal individuals may function to dampen responses to UV-induced endogenous Ag that are pathogenic in autoimmune disorders. However, these mechanisms might also facilitate the growth of UV-induced skin cancers.
Isolation of monoclonal antibodies specific for IL-4, IL-5, IL-6, and a new Th2-specific cytokine (IL-10), cytokine synthesis inhibitory factor, by using a solid phase radioimmunoadsorbent assay.
Authors: Mosmann T R , Schumacher J H , Fiorentino D F , Leverah J , Moore K W , Bond M W
Published: December 2022

Hybridomas were produced from a rat that was immunized with partially purified proteins from supernatants of induced Th2 cells. These preparations were enriched for cytokine synthesis inhibitory factor (CSIF, IL-10). The mAb in the supernatants were screened by a solid phase radioimmunoadsorbent assay using 35S-methionine-labeled secreted proteins from a lectin-stimulated Th2 clone. A total of 18 anticytokine mAb were isolated, comprising 6 anti-CSIF, 1 anti-IL-4, 1 anti-IL-5, and 10 anti-IL-6 mAb. The anti-CSIF mAb were separable into three groups. mAb in groups A and B neutralized and depleted bioactivity, and bound to overlapping but nonidentical subpopulations of CSIF molecules. The 2 mAb in group C did not neutralize CSIF activity, and bound to CSIF molecules not recognized by mAb from groups A or B. A two-site sandwich ELISA for CSIF could be established with the group A antibody, SXC1, combined with any of the three group B antibodies. The sensitivity of this assay was equivalent to that of the CSIF bioassay. These antibodies have been used to show that CSIF is responsible for most or all of the ability of Th2 supernatants to inhibit cytokine synthesis by Th1 cells. In addition, the ELISA has been used to confirm that CSIF is produced by Th2 but not Th1 clones.
Characterization of the CD45 molecule on murine intestinal intraepithelial lymphocytes.
Authors: Goodman T G , Chang H L , Esselman W J , LeCorre R , Lefrancois L
Published: December 2022

The CD45 molecule was analyzed from murine intestinal intraepithelial lymphocytes (IEL). Immunofluorescent staining of CD8+ IEL revealed varying degrees of reactivity with mAb specific for CD45-restricted determinants, some which are typically expressed only by B cells. Immunoprecipitation of CD45 molecules from IEL yielded an array of proteins with apparent (m.w.) ranging from 180,000 to 260,000. The m.w. 260,000 form was restricted to IEL, was distinct from the B220 molecule, and was the only CD45 isoform that expressed the CD45-associated carbohydrate differentiation Ag CT1. Moreover, the CT1 determinant was present on cells of the Thy-1- but not the Thy-1+ IEL subset. Sequential immunoprecipitation studies indicated that expression of the m.w. 260,000 protein was not restricted to CT1+ cells. The protein composition of the m.w. 260,000 CD45 isoform was examined by using the polymerase chain reaction for analysis of CD45 variable exon usage. In contrast to B cells in which the major CD45 mRNA contained all three variable exons (exons 4, 5, and 6), IEL CD45 mRNA contained significant amounts of two-exon, single exon, and zero variable exon forms. Restriction enzyme analysis identified the single exon form as exon 5 and the two-exon form as a mixture of exons 4 and 5 and exons 5 and 6. Metabolic labeling of CD45 in pulse-chase experiments suggested that the generation of this high m.w. protein was caused by post-translational modifications, perhaps glycosylation. Overall, the results indicated that the high m.w. form of CD45 and the addition of the CT1 determinant were generated via IEL-specific post-translational modifications and not by novel alternate exon usage.
Two families of monoclonal antibodies to alpha(1----6)dextran, VH19.1.2 and VH9.14.7, show distinct patterns of J kappa and JH minigene usage and amino acid substitutions in CDR3.
Authors: Wang D N , Chen H T , Liao J , Akolkar P N , Sikder S K , Gruezo F , Kabat E A
Published: December 2022

Nine groove-type mAb to alpha(1----6)dextran were cloned and sequenced. Together with previous reports from this laboratory, the VH and VL of 34 mAb have been sequenced, in which 10 VH19.1.2 and 11 VH9.14.7 combined with the V kappa-Ox1 gene to form two major families of anti-alpha(1----6)dextrans. The same D minigene (DFL16) was used by all VH19.1.2 and VH9.14.7 mAb; however, the patterns of JH and J kappa usage are quite different. VH19.1.2 mAb used only JH3 and J kappa 2, whereas VH9.14.7 mAb used three JH (JH1, JH2, and JH3) and all four active J kappa (J kappa 1, J kappa 2, J kappa 4, and J kappa 5). Relative uniformity in the lengths of VH CDR3 and the junctional sequences is seen in both families. Some mAb from different mouse strains share common structural features. The differences in idiotypic specificities and in the amino acid sequences suggest that VH19.1.2 and VH9.14.7 may differ in the conformation of CDR1 and CDR2. Combining with V kappa-Ox1 gene to generate groove-type combining sites to the single site-filling epitope of alpha(1----6)dextran, the two VH chains may require certain conformations of CDR3. Whether such conformational requirements influence the choice of J minigenes, the selection of the length of VH CDR3 and the sequences at junctions, are discussed.
Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha.
Authors: Aida Y , Pabst M J
Published: December 2022

When human neutrophils are incubated with LPS, they become primed for enhanced release of O2- in response to stimulation by FMLP. We investigated two aspects of LPS priming: 1) whether priming depends on secretion of TNF-alpha by monocytes present in neutrophil preparations, and 2) whether plasma is required for priming. Using plasma-Percoll gradients, we isolated neutrophils that contained only 0.1% monocytes. At 37 degrees C, these neutrophils were significantly primed by LPS (100 ng/ml) within 30 min. In contrast, LPS-treated monocytes required 60 min to secrete significant neutrophil-priming activity, the major component of which was TNF-alpha. Further, antibody against TNF-alpha failed to inhibit priming of neutrophils by LPS at 15, 30, and 45 min, and inhibited only 15% at 60 min. The results suggested that TNF-alpha or other factors from monocytes were not essential for priming of neutrophils by LPS. Neutrophils that had been washed free of plasma by centrifugation through 50% Percoll responded only weakly to LPS with respect to priming for enhanced O2- release and increased expression of alkaline phosphatase activity on the cell surface. Priming of washed neutrophils could be restored by adding back plasma (0.1 to 1.0%). This effect of plasma was not blocked by heating the plasma to 56 degrees C but was blocked at 100 degrees C. LPS priming could be blocked by polymyxin B, even in the presence of plasma. Thus, priming required both LPS and plasma. Neutrophils incubated with LPS in the absence of plasma were not primed by subsequent addition of plasma, but were primed by addition of plasma and LPS. Culture supernatants from neutrophils incubated with 20 ng/ml LPS in the absence of plasma failed to prime fresh neutrophils, but supernatants from neutrophils incubated with LPS in the presence of 1% plasma were able to prime fresh neutrophils. These results implied that neutrophils inactivated LPS and that plasma protected LPS from inactivation. Nevertheless, such inactivated LPS retained the ability to gel Limulus lysate at 10 pg/ml, and the ability to prime monocytes at 100 pg/ml. Thus, plasma prevented a neutrophil-specific inactivation of LPS.
Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc gamma receptor-specific mechanisms.
Authors: Crockett-Torabi E , Fantone J C
Published: December 2022

Signal transduction initiated by interaction of immune complexes (IC) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied by investigating the capacity of well-defined complexes to stimulate O2- generation in neutrophils. IC consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complexes) and at five times Ag excess (soluble complexes). Stimulation of human neutrophils with soluble and insoluble IC caused a dose-dependent activation of the respiratory burst and O2- generation. Incubation of neutrophils with cytochalasin B significantly enhanced O2- generation in neutrophils stimulated with soluble IC. In contrast, cytochalasin B treatment had a minimal effect on O2- generation in neutrophils stimulated with insoluble IC. Treatment of neutrophils with PGE1 or pertussis toxin (PTx) significantly inhibited O2- generation by soluble IC-stimulated neutrophils. However, neither PGE1 nor PTx treatment significantly altered O2- generation in neutrophils stimulated with insoluble complexes. Although O2- generation induced by soluble IC was significantly inhibited by mAb against both Fc gamma RII and Fc gamma RIII receptor, insoluble IC stimulation of neutrophil O2- generation was significantly diminished only by mAb against Fc gamma RIII receptor. Cross-linking of either Fc gamma RII or Fc gamma RIII receptors on neutrophil surfaces induced O2- generation, and this activation was inhibited by both PGE1 and PTx treatment. These findings indicate that soluble and insoluble ICs induce O2- production in human neutrophils through distinct mechanisms. Soluble IC induce activation of neutrophils through a PTx- and PGE1-sensitive pathway that is dependent upon both Fc gamma RII and Fc gamma RIII receptors. Although insoluble IC induce O2- production through a PTx and PGE1 insensitive pathway mediated primarily through Fc gamma RIII receptor.
Immunogenicity of a Haemophilus influenzae polysaccharide-Neisseria meningitidis outer membrane protein complex conjugate vaccine.
Authors: Donnelly J J , Deck R R , Liu M A
Published: December 2022

Polysaccharide-protein conjugate vaccines made with different carriers vary in their ability to elicit antipolysaccharide IgG antibody responses in young infants and an adult mouse model, suggesting that the carrier proteins used in the conjugate vaccines differ in their ability to act as carriers, or that additional mechanisms of immunogenicity play a role. A conjugate vaccine of Haemophilus influenzae PRP coupled to the outer membrane protein complex (OMPC) of Neisseria meningitidis serogroup B is immunogenic in children as young as 2 mo of age and is immunogenic in infant rhesus monkeys, an animal model for infant humans. In the present study, PRP-OMPC was found to induce efficient IgM to IgG switching of anti-PRP serum antibody in adult mice, whereas PRP conjugated to two other protein carriers did not. Thus the PRP-OMPC conjugate was examined in order to determine why PRP coupled to OMPC was so immunogenic, even more immunogenic than conjugates made with other carrier proteins. The OMPC carrier differs from the other protein carriers in that the proteins are present in a liposomal form containing lipids (including LPS) derived from the outer membrane of N. meningitidis. We studied the OMPC to see whether the different components or the nature of the OMPC carrier could contribute to its enhanced immunogenicity. Specifically we evaluated the OMPC for both classic Th cell carrier activity and adjuvanticity, and the LPS component of OMPC for systemic polyclonal B cell activation. Carrier recognition of the OMPC moiety of PRP-OMPC was demonstrated. In addition the PRP-OMPC conjugate vaccine was observed to have adjuvant properties for both T cell-dependent and T cell-independent Ag in the absence of LPS-induced systemic polyclonal B cell activation. These observations suggest that in addition to functioning as a classic protein carrier whereby the proteins in OMPC provide Th cell epitopes, the OMPC also has adjuvant activity that distinguishes it from other protein carriers and may contribute to the increased immunogenicity of PRP-OMPC conjugates in animal models.
Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.
Authors: Selvey L A , Tindle R W , Geysen H M , Haller C J , Smith J A , Frazer I H
Published: December 2022

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.
Aberrant expression of cytokine genes in peritoneal macrophages from mice infected with LP-BM5 MuLV, a murine model of AIDS.
Authors: Cheung S C , Chattopadhyay S K , Hartley J W , Morse H C , Pitha P M
Published: December 2022

Mice infected with LP-BM5 murine leukemia virus (MuLV) develop a syndrome denoted as murine AIDS. Macrophages harvested from the peritoneal cavities of these mice at 4 or 9 wk postinoculation with LP-BM5 MuLV were analyzed by Northern hybridization for the presence of the defective LP-BM5 virus and their ability to synthesize various cytokines upon induction with Newcastle disease virus (NDV) or (LPS). Neither IFN-alpha or IFN-beta was found to be constitutively expressed in LP-BM5-infected macrophages and in NDV induction studies, and the levels of biologically active IFN-alpha and its mRNA were found to be lower in LP-BM5 MuLV-infected macrophages than in the macrophages from uninfected controls. Similarly, after NDV or LPS induction, the levels of TNF mRNA and TNF protein were significantly lower in LP-BM5-infected macrophages than in macrophages from uninfected mice. The LP-BM5 MuLV-infected macrophages constitutively expressed low levels of IL-1 beta, and when induced with LPS, the relative levels of IL-1 beta were significantly higher in infected than in uninfected macrophages. Although no constitutive expression of IL-6 was detected, the levels of IL-6 mRNA induced with NDV were higher in LP-BM5 MuLV-infected macrophages than in controls. Thus, we found alterations in the expression of selected cytokines in macrophages from mice inoculated with LP-BM5 MuLV rather than a general deregulation of all cytokine expression. These results show that macrophages infected with the defective LP-BM5 virus respond differently to NDV- or LPS-stimulation and suggest that aberrant expression of certain cytokine genes may play a role in the immunopathologic condition in mice with murine AIDS.
Cellular and metabolic requirements for induction of macrophage procoagulant activity by murine hepatitis virus strain 3 in vitro.
Authors: Chung S , Sinclair S , Leibowitz J , Skamene E , Fung L S , Levy G
Published: December 2022

The cellular basis for the variation in induction of monocyte procoagulant activity (PCA) by murine hepatitis virus strain 3 (MHV-3) was examined using a set of recombinant inbred strains of mice derived from the resistant (A/J) and susceptible C57B1/6J (B) progenitors. Induction of PCA by MHV-3 required live virus and host protein and RNA synthesis. Absolute restriction for induction of PCA was observed at the level of the macrophage. Peritoneal macrophages from resistant parental A/J and RI strains (AXB5) could not be induced to express PCA when stimulated by MHV-3 alone or in the presence of lymphocytes from susceptible and H-2 compatible RI mice (AXB3) although they did respond to endotoxin (LPS). In contrast, macrophages from both susceptible (AXB3) and semisusceptible (AXB1) RI strains of mice expressed a similar increase in PCA after stimulation with MHV-3 in the absence of lymphocytes. The levels of PCA expressed by macrophages in the presence of Thy-1.2+ lymphocytes correlated with susceptibility to disease. Thy-1.2+ lymphocytes from susceptible RI AXB3 mice could induce levels of PCA in macrophages from semisusceptible RI AXB1 mice equivalent to that seen in cultures of macrophages and lymphocytes from susceptible mice. Further subfractionation of Thy-1.2+ cells demonstrated that L3T4+ cells instructed macrophages to produce PCA. Thy-1.2+ cells from MHV-3 immunized resistant AXB5 mice, but not from non-immunized mice, were able to suppress induction of PCA. This suppressor cell activity could be detected 4 days after immunization, reaching maximal activity at day 7 with significant suppression even at 28 days. The PCA was shown to have direct prothrombin cleaving activity (prothrombinase) by ELISA and immunofluorescence staining using the mAb 3D4.3. These results demonstrate that induction of a unique PCA (prothrombinase) is restricted at the level of the macrophage and define a regulatory role for T lymphocytes in its induction.
Analysis of the permissive association of a malaria T cell epitope with DR molecules.
Authors: Kilgus J , Jardetzky T , Gorga J C , Trzeciak A , Gillessen D , Sinigaglia F
Published: December 2022

In this study we examined the association of a promiscuous malaria T cell epitope, CS.T3, to different HLA-DR alleles. A large series of singly substituted or truncated variants of CS.T3 was prepared and tested for the ability to be recognised in association with, or to bind to, three distinct HLA-DR alleles (DR1, DRw11, and DRw14(w6)) and three natural variants of HLA-DRw11. We found that although association with the different DR molecules mapped to identical or closely overlapping regions of the peptide, distinct substitutions could drastically influence the capacity of the peptide to interact with one but not another of the three DR molecules tested. Based on analysis of the distribution of residues recognized by T cell clones restricted to the different DR alleles, we suggest that the peptide CS.T3 is not bound, at least for the three DR examined, as an alpha-helix. In addition we tested three subtypes of DRw11 as APC for the CS.T3 analogues and observed that the peptide is most likely bound in the same conformation to the three natural variants of the DRw11 molecule.
A shared kappa reciprocal fragment and a high frequency of secondary Jk5 rearrangements among influenza hemagglutinin specific B cell hybridomas.
Authors: Clarke S , McCray S
Published: December 2022

Ig kappa-chain gene rearrangement results in the displacement or loss of the DNA immediately 5' of Jk. This retained DNA is found on a different size fragment than in the germline (a reciprocal fragment), and contains the reciprocal joint of rearranged Vk and Jk genes, the back-to-back fusion of the heptamer/nonamer recombination signals. B cells of independent origin rarely have reciprocal fragments of the same size. However, we report that 9 of 15 B cell hybridomas of independent origin have reciprocal fragments of the same size (8-kb BamHI fragments) unrelated to their productive rearrangements. An 8-kb reciprocal fragment has also occurred on about 25% of the kappa alleles of normal splenic B cells. We find that the reciprocal fragments in two of these hybridomas contain the reciprocal joints of Jk1 genes and different Vk8 genes. In addition, we find that at least 8 of the 12 Jk4 or Jk5 expressing hybridomas have undergone double recombinations on their productive kappa alleles. The implications of these findings on the high frequency of 8-kb reciprocal rearrangements and on Vk rearrangement are discussed.
Cytolytic lymphocytes induce both apoptosis and necrosis in target cells.
Authors: Zychlinsky A , Zheng L M , Liu C C , Young J D
Published: December 2022

We examined the role of programmed cell death (apoptosis) in killer lymphocyte-mediated cytotoxicity. Two parameters of cell death, 51Cr release and DNA fragmentation, were assayed. Lymphokine-activated killer cell- or CTL-mediated death was inhibited in target cells where transcription or translation were blocked. Dying target cells showed ultrastructural changes typically associated with both apoptosis and necrosis. In contrast, target cells pretreated with macromolecular synthesis inhibitors and incubated with lymphokine-activated killer cells showed morphologic signs of necrosis only. Zn2+, an inhibitor of endonucleases, inhibited DNA fragmentation, but not 51Cr release in YAC-1 target cells, suggesting that the two effects can be dissociated. Finally, the cytotoxic effect of perforin, a pore-forming protein of killer lymphocytes that is known to cause necrotic death, was unaffected by the inhibition of either RNA or protein synthesis in target cells. Taken together, these results suggest that killer lymphocytes can induce both necrosis and apoptosis and that the two types of death can be dissociated with specific inhibitors.