bluuebunny/crossref_metadata_2025 huggingface dataset:
Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal transduction pathways.
Authors: Welham M J , Schrader J W
Published: December 2022

The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.
In vitro growth of bone marrow-resident T cell precursors supported by mast cell growth factor and IL-3.
Authors: Chervenak R , Dempsey D , Soloff R S , Smithson G
Published: December 2022

The growth requirements of bone marrow-resident cells that are able to differentiate along the T cell lineage (pre-T cells) have not been well established. We recently have shown that the T cell-derived lymphokine IL-3 is able to maintain pre-T cells in vitro for at least 2 weeks. However, in our initial studies, we were not able to ascertain whether IL-3 induced pre-T cell growth during culture, or whether IL-3 simply maintained the viability of these progenitors. To address this issue, we used a multiple dose assay system to assess the level of pre-T cell activity (thymic repopulation) in a selected population of bone marrow cells (CD3-, Thy-1.2+) both before and after culture in IL-3. In addition, we tested the potential role of mast cell growth factor (MGF) in the growth and maintenance of pre-T cells in vitro. The results of these studies showed that IL-3 produced a modest, but consistent increase in the pre-T cell activity during culture. Culture of CD3-, Thy-1.2+ bone marrow cells in MGF also resulted in an increase in the total amount of detectable pre-T cell activity among the cultured cells. The most dramatic increases in pre-T cell activity, however, were induced by the culture of the selected marrow cells in both MGF and IL-3. Cultures supplemented with both cytokines produced net increases in pre-T cell activity of 40- to 75-fold after 10 days of culture. Because the increases in pre-T cell activity were not accompanied by observable increases in the size of thymic colonies produced by the pre-T cells, the increased levels of pre-T cell activity appeared to result from increases in pre-T cell numbers during culture. Thus, in addition to the other activities ascribed to MGF, this cytokine displays pre-T cell growth factor activity and can synergize with IL-3 in that capacity. The use of MGF in conjunction with IL-3 provides the best system described to date for the propagation of pre-T cells in primary bone marrow cell cultures.
Predominance of Th1 cells in ocular tissues during herpetic stromal keratitis.
Authors: Niemialtowski M G , Rouse B T
Published: December 2022

Herpetic stromal keratitis (HSK) appears to represent an immunopathologic response in the cornea of the eye to HSV-1. T cells of the CD4+ subset were shown to be involved in the mediation of HSK, but how they subserve an immunopathologic role is uncertain. In the present report, we have isolated cells from eyes in the active phase of HSK and studied their cytokine profile after culture in vitro or stimulation with Ag or nonspecific mitogens. Inflammatory cells recovered from eyes consist of polymorphonuclear leukocytes, macrophages, and lymphocytes. As reported before, all the lymphocyte recovered were of the CD4+ phenotype. After stimulation in vitro with Ag or mitogen the cytokines IL-2, IFN-gamma, and TNF-alpha/beta were produced, but not the cytokines IL-4 and IL-10. Thus, on the basis of cytokine profile, ocular lymphocytes were identified as Th1 cells. Ocular cells were also stimulated with PMA and shown to produce IL-1. The results were discussed in terms of the possible means by which the Th1 cells induce tissue damage in HSK as well as in terms of the possible means by which a preferential accumulation of Th1 cell occurs in the eye.
Cytokine expression in vivo during murine listeriosis. Infection with live, virulent bacteria is required for monokine and lymphokine messenger RNA accumulation in the spleen.
Authors: Poston R M , Kurlander R J
Published: December 2022

To examine the regulation of cytokine synthesis during murine listeriosis, we have monitored IFN-gamma, TNF-alpha, and IL-1 beta mRNA levels in the spleens of C57B1/6 mice after the i.v. infusion of virulent and nonvirulent preparations of Listeria monocytogenes (LM). Messenger RNA coding for TNF, IL-1, or IFN did not become detectable until approximately 12 to 15 h after the infusion of virulent LM. Levels of each cytokine mRNA then increased synchronously reaching peak or near peak levels around 24 h after infection. Levels gradually decreased over the next 4 to 5 days. Unlike virulent LM, neither heat-killed LM, nor nonvirulent LM variants lacking listeriolysin O, stimulated monokine or IFN mRNA accumulation even when administered in very large doses. To gain perspective concerning the response to LM, we examined the early pattern of cytokine mRNA accumulation induced by Salmonella typhimurium (ST), an intracellular pathogen expressing LPS. We noted at least three significant differences between the cytokine responses to LM and ST: 1) monokine mRNA levels increased much more rapidly (within 1 h) after ST infection; 2) unlike LM, ST retained the capacity to stimulate cytokine mRNA production when injected as heat-killed bacteria; 3) in contrast to LM, ST could not trigger the early IFN production characteristic of LM infection. Our data suggest that monokine and IFN production early in listeriosis are critically linked with the process of bacterial invasion of host cells. The timing and pattern of cytokine mRNA accumulation in this setting is qualitatively different from that induced by LPS. The pathway described in these studies may also play a role in the host cytokine response to other intracellular pathogens as well.
Lipopolysaccharide induces Egr-1 mRNA and protein in murine peritoneal macrophages.
Authors: Coleman D L , Bartiss A H , Sukhatme V P , Liu J , Rupprecht H D
Published: December 2022

Bacterial LPS has diverse effects on the function of immune cells, in general, and macrophages, in particular. The intracellular molecular events that mediate the effects of LPS are unclear. We undertook a series of studies in thioglycollate-elicited murine peritoneal macrophages to evaluate the effect of LPS on expression of Egr-1, a member of the immediate early response gene family. Egr-1 may function as an intranuclear "third messenger" because it is rapidly induced in a variety of cell types and encodes a 75- to 80-kDa nuclear phosphoprotein that activates transcription of genes containing the DNA consensus sequence GCGGGGGCG. LPS from Salmonella minnesota Re595 induced a maximal increase in Egr-1 mRNA in macrophages after 30 to 60 min of incubation that returned to baseline level by 120 min. LPS increased Egr-1 mRNA at 0.01 to 0.1 ng/ml with a maximal effect at 10 to 100 ng/ml. LPS markedly increased the transcription rate of Egr-1 by 10 min of incubation using nuclear run on analysis. Using a polyclonal anti-Egr-1 antibody, nuclear staining for Egr-1 protein was prominent after 1 to 2 h of incubation with LPS and declined to baseline by 4 h. Inasmuch as protein kinase C (PKC) has been implicated in mediating the effects of LPS, we determined whether PKC was required for LPS to increase Egr-1 mRNA. Two pharmacologic approaches were used to deplete PKC, PMA pretreatment, and H-7. The induction of Egr-1 mRNA by LPS was markedly reduced in PKC-depleted macrophages. These data reveal that LPS induces transcriptional activation of Egr-1 and increases Egr-1 protein in peritoneal macrophages. In addition, these findings support further study of the potential role of Egr-1 in mediating the effects of LPS in peritoneal macrophages.
Evidence for early onset, polyclonal activation of T cell subsets in mice homozygous for lpr.
Authors: Giese T , Davidson W F
Published: December 2022

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the accumulation of two functionally anergic T cell subsets, a predominant B220+CD4-CD8- double negative (DN) population and a minor, closely related CD4 dull+ B220+ population. Lymph nodes from diseased lpr and gld mice also contain abnormally high numbers of conventional T cells, and we reported recently that a high proportion of lpr and gld CD4+B220- T cells have the hallmarks of primed or memory T cells. In the present study, we further investigated the extent, ontogeny, and possible causes of T cell activation in lpr and gld mice. The criteria used to identify primed or memory T cells included activation-dependent increases in the expression of CD44, LFA-1, and the early activation Ag, CD69, and decreases in the expression of Mel-14 and CD45RB, as well as quantitative differences in the in vitro production of IFN-gamma and the TNF-alpha by stimulated cells. A comparison of TCR V beta gene utilization by lpr T cell subsets also was undertaken. The results showed that T cell activation was widespread and complex. CD8+ T cells exhibited a similar pattern of activation to CD4+B220- T cells. The activation of these two subsets occurred in parallel, was in evidence by 4 to 6 wk of age, and was both chronic and progressive. The proportions of CD44hiLFA-1hi, CD4+B220-, and CD8+ T cells increased steadily between 4 and 20 wk of age, but changes in T cell growth, Mel-14, and CD45RB expression and cytokine secretion were not observed until mice were older than 11 wk. A very different pattern of activation was observed for B220+ T cells. At all ages, B220+ DN and CD4+B220+ T cells were CD44hiMel-14hi and 60 to 75% were CD69+. The expression of CD69 appeared to be stimulus dependent rather than constitutive, suggesting that these cells, too, may be chronically stimulated in vivo. In keeping with their anergic state, DN T cells responded poorly to cross-linking of CD69. The stimuli inducing chronic activation of CD4+B220- and CD8+ T cells are unlikely to include inappropriate reactions to autoantigens because there was no evidence for selective accumulation of CD4+ or CD8+ T cells bearing particular V beta genes or potentially self-reactive cells that normally are deleted in the thymus. By comparison, C3H-lpr DN cells displayed some potentially significant differences in V beta 6 and V beta 9 expression from CD4+B220- and CD8+ T cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigenic Properties of Euglena Gracilis
Authors: Elmore Mary E.
Published: December 2022

The problem of the antigenic properties of unicellular chlorophyll-bearing organisms was suggested by the work of Sherwood (1) in which he attempted to immunize rabbits to the chloroplasts of several higher plants. The serum of the treated animals did not bring about release of the chlorophyll from the chloroplasts in the presence of fresh serum or produce any changes detectable microscopically, and agglutinin production was inconstant. Moreover, in extensive unpublished experiments with purified colloidal chlorophyll, he found no evidence of antibody formation. It was thought that a thorough immunological study of some simple chlorophyll-bearing organism might further our knowledge of the antigenic properties of chlorophyll in the chloroplast and, in addition, give valuable information about a group which has been insufficiently studied from this standpoint.

Rosenblat-Lichtenstein (2), using pure cultures of several species of Chlorophyceae, showed that specific agglutinating sera of moderately high titer could be produced.
Studies in Specific Hypersensitiveness
Authors: Clarke J. A. , Donnally H. H. , Coca A. F.
Published: December 2022

Statistical evidence is presented supporting the conclusion that the atopic shock tissues (bronchial and upper respiratory) are subject to separate hereditary influence.
Some Properties of Two Active Substances Contained in Certain Scarlatinal Streptococcus Filtrates
Authors: Hooker Sanford B. , Follensby Edna M.
Published: December 2022

Some strains of scarlatinal streptococci produce at least two highly active substances which appear to be toxins because they evoke the production of antibodies which neutralize according to the law of multiple proportions. Toxin “A” has not been found to differ from toxins produced by other strains of scarlatinal streptococci. Toxin “B” is active in only a few individuals, who may be negative to “A,” and differs in other respects: It is thermolabile, is less stable in storage, is more easily dialyzed, and is precipitated by lower concentrations of ammonium sulfate or sodium chlorid than toxin “A.” It does not appear to produce a binding toxoid; it is produced somewhat earlier than “A” in broth but less is formed in a simple synthetic medium.

Neither “A” nor “B” is adsorbed by moderate amounts of kaolin; their corresponding antitoxins are not adsorbed by washed homologous cocci.
Lymphokine-activated killer cells. VII. IL-4 induces an NK1.1+CD8 alpha+beta- TCR-alpha beta B220+ lymphokine-activated killer subset.
Authors: Ballas Z K , Rasmussen W
Published: December 2022

Murine IL-2-induced lymphokine-activated killers (LAK) can be divided into two mutually exclusive subsets: NK1.1+CD8- and NK1.1-CD8+. We have previously established that the lytically active subset of each of these two populations expressed B220, as determined by the mAb 6B2, on its surface. In this study, we examined the lytically active subsets induced by IL-4. We found that, similar to IL-2, the lytically active IL-4-LAK expressed B220 on their surface. Similar to IL-2-LAK, IL-4-LAK cultures also contained NK1.1+CD8- B220+, and NK1.1-CD8+B220+ subsets. IL-4-LAK cultures, however, contained two novel and unexpected lymphocyte subsets determined by their surface markers to be either: i) NK1.1+CD8 alpha+beta-B220+, or ii) NK1.1-CD8 alpha+beta-B220+. These subsets were not derived from NK1.1+ or CD8+ precursors but were induced from a CD4-CD8-NK1.1-Slg- splenocyte subpopulation. Most of the CD8 alpha+beta- cells seen in the IL-4 cultures expressed TCR-alpha beta with little or no TCR-gamma delta detected on their surface. Similar to IL-2-LAK, the IL-4-LAK subsets appeared to display a bias as to their susceptible target cells with the NK1.1-CD8 alpha+beta+ subset being most potent against trinitrophenyl-modified autologous lymphoblasts (2,4,6-trinitrobenzene sulfonic acid (TNBS)-self). The NK1.1+CD8 alpha-beta- effectors were most potent against YAC-1 and CL27A with little activity against TNBS-self. All three targets were susceptible to lysis by the NK1.1+CD8 alpha+beta- subset. These findings document the characteristics of a novel lymphocyte subset, derived from splenic precursors, which shares certain features with cells previously thought to be present exclusively in intraepithelial lymphocytes.
Herpes simplex virus interferes with monocyte accessory cell function.
Authors: Hayward A R , Read G S , Cosyns M
Published: December 2022

HSV is a successful human pathogen that causes severe infections in immunocompromised adults and newborn humans. The possibility that HSV might evade host immune responses by interfering with accessory cell function was investigated in vitro using newborns' monocytes adhered to plastic. These cells lost their ability to present staphylococcal enterotoxin B to resting T cells in a stimulatory form after overnight culture with HSV. The interference with T cell proliferation required live virus and was abolished by heat- or UV inactivation. The T cell proliferative response was restored by the addition of IL-2. Furthermore, HSV-precultured monocytes had a reduced production of IL-1 alpha and TNF-beta after phorbol-ionomycin stimulation. Wild-type HSV and a HSV mutant lacking the primary virion host shutoff protein both interfered with IL-1 synthesis and presentation of staphylococcal enterotoxin B for T cell proliferation. These results suggest that HSV can interfere with the provision by human monocytes of costimulator factors that are essential for T cell stimulation. This effect of HSV may be due to secondary shutoff mechanisms that decrease host protein synthesis or secretion after HSV infection.
In vivo desensitization to leukotriene B4 (LTB4) in the rabbit. Inhibition of LTB4-induced neutropenia during intravenous infusion of LTB4.
Authors: Marleau S , Fortin C , Poubelle P E , Borgeat P
Published: December 2022

Bolus injections of leukotriene B4 (LTB4) at 30-min intervals repeatedly induced similar profound and reversible neutropenias. In contrast, after a 30-min infusion of LTB4, the neutropenia induced by bolus injections of LTB4 was inhibited in a dose-dependent manner; a threshold inhibition was seen at the infusion rate of 10 ng LTB4/min/kg, whereas almost complete inhibition was observed at 50 ng LTB4/min/kg. Steady state arterial plasma concentrations of LTB4 increased proportionally to LTB4 infusion rate, ranging from 0.15 +/- 0.01 nM (control) to 2.80 +/- 0.16 nM (100 ng/min/kg). Extending the infusion period of LTB4 up to 330 min did not result in an enhanced inhibition of the neutropenia in response to bolus injections of LTB4. Reversibility of the desensitization was shown by an almost complete recovery of the neutropenic response within 30 min after cessation of the infusion. The desensitization achieved towards LTB4 showed some specificity, inasmuch as a profound and reversible neutropenia was observed in response to a bolus of either FMLP or C5a under conditions in which sensitivity to LTB4 was lost. These findings suggest that a specific desensitization to LTB4 is feasible in vivo and may constitute a useful approach, in addition to the use of 5-lipoxygenase inhibitors and LTB4 antagonists, to delineate the significance of LTB4 as a mediator of inflammation.
Generation of diacylglycerol and ceramide during homologous complement activation.
Authors: Niculescu F , Rus H , Shin S , Lang T , Shin M L
Published: December 2022

Formation of sublytic terminal complement complexes (TCC) on nucleated cells produces transient increase in [Ca2+]i and activates protein kinase C. The present study is to evaluate whether TCC can generate endogenous signal messengers other than Ca2+ that regulate cell activities by measuring mass-levels of sn-1,2-diacylglycerol (DAG) and ceramide. As targets, lymphoblastoid human B cell lines JY25 and its mutant JY5 were used. JY5, cells deficient in glycosylphosphatidylinositol-anchored proteins with higher lytic susceptibility to human complement, are four times more efficient in forming C5b-9. When cells sensitized with limited anti-class II IgG were exposed to human serum to generate sublytic TCC, a sustained increase in DAG and ceramide was observed with a maximum 3.6-fold DAG increase over basal level in JY25 and 2.8-fold in JY5, and 6.3-fold ceramide increase in JY25 and 2.8-fold in JY5. The effect of TCC was evaluated with C7-deficient human serum (C7D) +/- C7 and also with C5b6, C7, C8, and C9 proteins. The DAG and ceramide increase by C7D + C7 over C7D control were 1.6- and 1.8-fold, respectively, in JY25, and 2.3-, and two-fold in JY5. TCC activation also induced an increased hydrolysis of sphyingomyelin and phosphatidylcholine. In addition, DAG increase by TCC was primarily achieved by C5b-7 and preincubation of cells with pertussis toxininhibited DAG increase, suggesting an involvement of a pertussis toxin-sensitive GTP-binding protein. As important signal transduction molecules, DAG and ceramide generated in response to TCC assembly, could participate in cell activation during inflammation and repair.
Depression of macrophage respiratory burst capacity and arachidonic acid release after Fc receptor-mediated phagocytosis.
Authors: Schwacha M G , Gudewicz P W , Snyder J A , Loegering D J
Published: December 2022

The phagocytosis of IgG-coated erythrocytes (ElgG) by macrophages results in a subsequent depression of macrophage phagocytic function, respiratory burst capacity, and bactericidal activity. Our study was carried out to determine the importance of impaired arachidonic acid release in the depression of the respiratory burst after ElgG phagocytosis. The depression of triggered H2O2 production after ElgG phagocytosis was not due to cyclooxygenase products because indomethacin or aspirin did not modify the depression. Further studies revealed that the depression of triggered H2O2 production after ElgG phagocytosis was associated with a depression in the ability of macrophages to release arachidonic acid in response to PMA, zymosan, or calcium ionophore. The addition of exogenous arachidonic acid partially prevented the depression of triggered H2O2 production after ElgG phagocytosis. Unlike phagocytosis mediated by FcR, complement receptor-mediated phagocytosis did not alter H2O2 production or arachidonic acid release. Ligation of FcR was not sufficient to depress triggered H2O2 production and arachidonic acid release because these functions were not depressed when phagocytosis was inhibited with cytochalasin D. Thus, it was found that the depression of triggered H2O2 production by macrophages after FcR-mediated phagocytosis was associated with impaired release of arachidonic acid and that H2O2 production could be partially restored by the addition of arachidonic acid. These results suggest that the impairment of arachidonic acid release after FcR-mediated phagocytosis contributes to the depression of macrophage respiratory burst capacity after FcR-mediated phagocytosis.
Human granulocytes express a 55-kDa lipopolysaccharide-binding protein on the cell surface that is identical to the bactericidal/permeability-increasing protein.
Authors: Weersink A J , van Kessel K P , van den Tol M E , van Strijp J A , Torensma R , Verhoef J , Elsbach P , Weiss J
Published: December 2022

Several LPS-binding proteins have been identified on the surface of human granulocytes (polymorphonuclear leukocyte (PMN)). We describe a plasma-membrane associated ca. 55-kDa LPS-binding protein of human PMN that is indistinguishable from the bactericidal/permeability-increasing protein (BPI). To detect LPS-binding proteins on the cell surface, PMN were biotinylated before detergent solubilization and incubation with LPS-coated beads. Several biotinylated proteins bound to LPS-coated beads but not to uncoated beads and were characterized after elution with detergent by SDS-PAGE and western blotting using streptavidin-horseradish peroxidase. The spectrum of biotinylated proteins binding to and eluting from LPS-coated beads increased as the number of beads incubated with PMN lysate increased. However, at all concentrations of beads a 55-kDa protein was a dominant component of the eluate. Binding of the 55-kDa protein to LPS-coated beads was inhibited by lipid A, and both homologous and heterologous LPS, but not by peptidoglycan. Similar amounts of biotinylated 55-kDa LPS-binding protein were detected on PMN from patients with paroxysmal nocturnal hemoglobinuria who lacked membrane bound CD14, a known ca. 55-kDa plasma membrane-associated LPS-binding protein, indicating that the recovered biotinylated protein is not CD14. Several pieces of evidence, however, do indicate that the 55-kDa surface protein is BPI: 1) flow cytometry of PMN after labeling with rabbit anti-BPI serum and FITC-labeled goat anti-rabbit IgG revealed immunoreactive surface molecules on resting PMN and, in increased amounts, on PMN stimulated with FMLP or TNF; 2) This antiserum specifically and quantitatively inhibited binding of the biotinylated 55-kDa species to LPS-coated beads; 3) both BPI and the 55-kDa protein migrated as a doublet during SDS-PAGE and were both converted to single migrated species after N-glycosidase F treatment; 4) chemical cleavage of the biotinylated protein and native BPI with N-chlorosuccinimide yielded the same fragments. Thus, we have positively identified BPI as a LPS-binding protein on the surface of PMN. The role of this potent antibacterial, endotoxin neutralizing protein on the surface of PMN remains to be established.
A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression.
Authors: Godfrey D I , Kennedy J , Suda T , Zlotnik A
Published: December 2022

We have subdivided mouse CD4-CD8-CD3- triple-negative (TN) thymocytes into four subsets based upon expression of CD44 and CD25, including CD44+CD25-, CD44+CD25+, CD44-CD25+ and CD44-CD25-. Characterization of these cells revealed several features distinct to each subset, in particular the expression of high levels of c-kit (the receptor for stem cell factor) by CD44+CD25-TN and CD44+CD25+TN but not by CD44-CD25+TN and CD44-CD25-TN. The CD44+CD25+TN subset also included the IL-7 and stem cell factor-responsive cells, whereas only minimal responsiveness was observed by the CD44- populations. These subsets also showed differential cytokine production potential (CD44+CD25- > CD44+CD25+ > CD44-CD25+ > CD44-CD25-) after stimulation with calcium ionophore, PMA and IL-1. The repopulation potential of these subsets in 2-deoxyguanosine-treated fetal thymic lobes supports the following maturation sequence: CD44+CD25- -->CD44+CD25+ -->CD44-CD25+ -->CD44-CD25-. Furthermore, the sequence of progression from CD44+CD25+ to CD44-CD25+ cells was confirmed by their TCR beta-chain gene configuration. The former population exhibits germ-line TCR beta-chain configuration, whereas the latter subset shows a rearranged pattern.
Adenovirus E1A inhibits IFN-induced resistance to cytolysis by natural killer cells.
Authors: Routes J M
Published: December 2022

Infection of target cells with cytopathic viruses inhibits IFN induction of cytolytic resistance to NK cell-mediated cytolysis [IFN-mediated cytoprotection (IFN-MCP)]. It has been thought that the virally induced inhibition of IFN-MCP is secondary to the shutdown of cellular macromolecular synthesis that accompanies cytopathic virus infections. Group C, adenovirus serotype 5 (Ad5) infection inhibits both IFN-MCP and cellular protein synthesis. This study determined if the Ad5-induced inhibition of IFN-MCP was independent of adenovirus (Ad) infection and secondary only to the expression of the Ad early region 1A gene (E1A). To test this hypothesis, 4-h NK cytolysis assays were performed on IFN-gamma-treated human cells infected with an Ad5 E1A deletion mutant, dl343, or transfected with the Ad5 E1A gene. IFN-MCP was not inhibited by infection with dl343, despite the production of large amounts of both early (E1B, p55) and late (hexon) Ad proteins. In contrast to E1A-negative, parental cell lines, IFN-MCP was blocked in Ad5 E1A-transfected epithelial and fibroblastic cell lines. Genetic mapping studies within the E1A gene demonstrated that expression of only the first exon of E1A was sufficient to inhibit IFN-MCP. DNA sequence homology of E1A genes between different Ad groups (group A, Ad12; group C, Ad5) is limited almost entirely to three conserved regions located within the first exon of E1A. Because IFN-MCP was also blocked in Ad12 E1A-transfected cell lines, expression of one or more of the E1A-conserved regions may be necessary to inhibit IFN-MCP. In summary, the expression of E1A gene products inhibited IFN-MCP independently of virus infection. E1A's inhibition of IFN-MCP has the net effect of promoting the selective NK cell-mediated clearance of Ad-infected or Ad-transformed human cells.
IFN-gamma, but not IL-4, synthesis by antigen-primed murine T cells is IL-2 dependent. Differential requirements in de novo and established responses.
Authors: Yang X , HayGlass K T
Published: December 2022

Ag-induced, CD4 T cell-dependent synthesis of IL-4 and IFN-gamma plays a critical role in the induction, maintenance, and regulation of allergic responses. The requirement for IL-2 and IL-4 in the initial induction of IL-4 T cell responses is well established. However, it remains unclear whether IL-2 is required for priming of IL-4 responses, IL-4 gene expression, or both. In this report, we dissociate the IL-2 dependence of developing and established IL-4 responses. The capacity of spleen cells from naive and allergen-immunized mice to synthesize IL-4 after Ag-specific vs polyclonal restimulation is examined in overnight cultures. Although de novo induction of IL-4 and IFN-gamma production obtained after in vitro activation of cells from naive animals with immobilized anti-CD3 is IL-2 dependent, Ag-driven IL-4 synthesis by CD4 T cells from OVA (alum)- or ragweed extract (alum)-immunized mice is independent of IL-2. The data demonstrate the IL-2 independence of CD4 T cell-derived IL-4 synthesis by allergen-primed cells, distinguishing IL-2-dependent from IL-2-independent stages of IL-4 responses. In contrast to the finding that IL-2 is not required for IL-4 gene expression by primed T cells in allergen-driven responses, both induction and expression of IFN-gamma responses are strictly IL-2 dependent. This differential requirement for IL-2 in IL-4 and IFN-gamma gene expression may be significant in the maintenance of IL-4-dominated responses in allergic individuals.
A novel cellular model (SPGM 1) of switching between the pre-B cell and myelomonocytic lineages.
Authors: Martin M , Strasser A , Baumgarth N , Cicuttini F M , Welch K , Salvaris E , Boyd A W
Published: December 2022

The suspension pro granulocyte/macrophage (SPGM1) cell line was established from a transplantable mouse progranulocytic/promacrophage tumor. Surprisingly, SPGM1 cells expressed a typical CD5 pre-B cell phenotype, being positive for Ly-1 (CD5), PB76, B220 (CD45RA), and the pre-B Ig receptor complex (microH chains, lambda 5 and vpre-B surrogate L chains, and the IgM alpha (mb-1) and Ig beta (B29) co-receptor molecules). Southern Blot analysis revealed clonal rearrangement of the microH chain locus and germ-line L chain loci. SPGM1 formed blast cell-, macrophage-, and occasional granulocytic colonies in soft agar in the presence of murine IL-3. IL-3 also induced macrophage differentiation of SPGM1 cells in suspension cultures. The earliest changes were detectable at 24 h by Northern blot analysis. IL-3-treatment increased Mac1 mRNA, induced c-fms mRNA, and down-regulated mRNA for mu, lambda 5, vpre-B and mb-1. After 2 to 4 days the cells were larger, strongly adherent, expressed the macrophage markers Mac1 and F4/80, had lost microH chain and PB76 surface expression, and readily phagocytosed latex beads. Thus SPGM1 has all the characteristic features of a CD5+ pre-B cell line. However, IL-3 predominantly induced SPGM1 to switch its differentiation program from a pre-B cell to a macrophage. This inducible, rapid switch of virtually the entire population provides a unique model for the molecular analysis of mechanisms involved in cell-fate determination.
A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments.
Authors: Reed G L , Kussie P , Parhami-Seren B
Published: December 2022

Streptokinase (SK), a bacterial product of pathogenic Streptococcus species, is now widely used as an effective therapy for the treatment of heart attacks. Because naturally occurring antibody to SK is ubiquitous, serious allergic reactions to SK therapy are common. To begin to identify regions of the molecule that are important for the antigenicity of SK we performed studies using a panel of 51 hybridomas producing anti-SK antibodies, recombinant SK fragments, and assays of SK activity. Antibodies generated from mice hyperimmunized with wild-type SK were shown to fall into six distinct complementation groups by competitive binding studies. Recombinant SK fragments were used to determine the peptide regions recognized by these complementation groups. Correlation of the effects of the mAb on SK function, with knowledge of their SK fragment-binding pattern, suggested regions of the SK molecule that are important for the construction and the catalytic function of the SK-plasminogen activator complex.
Increased protein kinase C activity in low density eosinophils.
Authors: Bates M E , Bertics P J , Calhoun W J , Busse W W
Published: December 2022

Eosinophil heterogeneity is expressed in cell density, membrane receptors and function. It has been observed that increases in some functional activities correlate with decreased sedimentation density in human eosinophils. However, the cellular mechanisms to explain the up-regulation of eosinophil function have not been fully explored. Protein kinase C (PKC) is an important family of enzymes mediating signal transduction for a wide variety of functions in many different cell types. Changes in the activity of PKC could explain some of the observed differences in function. In these experiments, PKC activity of human granulocyte lysate supernatants was measured as the phosphatidyl serine-dependent transfer of 32P from [gamma-32P]ATP to a protein substrate under conditions of maximal stimulation; a measure of activatable PKC concentration. We observed that the activity present in eosinophils (87.2 +/- 8.4 pmol PO4 incorporated into histone per minute per 10(6) cells, n = 30) was not significantly different from that of neutrophils assayed under the same conditions (91.5 +/- 5.6 U, n = 31) but the percent of total activity that was phosphatidyl serine dependent was greater in eosinophils (97 +/- 1% vs 81 +/- 1% for neutrophils, p = 0.001). Blood eosinophils isolated from low density Percoll fractions had a higher activity (120 +/- 16 U) than that found in the higher density cells from the same subjects (81 +/- 19 U, n = 9, p = 0.011). When eosinophils recovered from bronchoalveolar lavage (BAL) fluid after segmental Ag challenges were assayed, the PKC activity of BAL eosinophils was similar to that of blood-derived eosinophils of equal density and low density BAL eosinophil PKC tended to be equal to or greater than higher density cells. The beta isozyme of PKC but not the alpha or gamma was detected in eosinophils by Western blotting with isozyme-specific mAb. These data indicate that eosinophil PKC activity is primarily caused by the beta-isozyme, is related to cell density in blood-derived cells, and may have a relationship to cell function.
Enhanced degradation of messenger RNA encoding myelin proteins by terminal complement complexes in oligodendrocytes.
Authors: Shirazi Y , Rus H G , Macklin W B , Shin M L
Published: December 2022

Sublytic terminal C complexes (TCC) are capable of stimulating cells and affect the target cell activity. Activation of TCC that generates leukotriene B4 in oligodendrocytes, the myelin-forming cells of the central nervous system, is also a required process in antibody-mediated demyelination of rodent cerebellar explants. In the present study, the effect of TCC on myelin protein gene expression was studied in primary rat oligodendrocytes in culture. Sublytic activation of serum C reduced accumulation of mRNA encoding proteolipid protein (PLP) and myelin basic protein (MBP) within 1 h, but not beta-actin mRNA. C activation, on the other hand, induced sustained expression of c-jun mRNA. Experiments using C7-deficient human serum to determine the role of TCC showed that selective MBP and PLP mRNA down-regulation was achieved only when C7 was reconstituted to form TCC. The C7 requirement was also observed in the presence of alpha-amanitin. Post-transcriptional regulation was explored by determining mRNA decay, which demonstrated that the MBP and PLP mRNA were selectively destabilized when C7 was reconstituted. Limited exploration of the signals responsible for the TCC effect revealed that down-regulation of mRNA by TCC was significantly influenced by Ca2+ on PLP, whereas MBP did not show the same Ca2+ sensitivity as PLP. The TCC-mediated MBP mRNA decay was completely abrogated by HA1004, an inhibitor for the cAMP- and cGMP-dependent protein kinases, but not by H7, a protein kinase C inhibitor.
Effect of vasoactive intestinal peptide, somatostatin, and substance P on spontaneous IgE and IgG4 production in atopic patients.
Authors: Kimata H , Yoshida A , Fujimoto M , Mikawa H
Published: December 2022

The effect of vasoactive intestinal peptide (VIP), somatostatin (SOM), and substance P (SP) on spontaneous human IgE and IgG4 production in atopic patients was studied. In cultures of mononuclear cells (MNC), VIP inhibited both IgE and IgG4 production without affecting IgM, IgA, IgG1, IgG2, or IgG3 production. In contrast, SOM inhibited only IgE production whereas SP inhibited only IgG4 production without affecting production of other isotypes or other IgG subclasses. The effect of neuropeptides was specific because each was specifically blocked by a corresponding neuropeptide antagonist. To achieve the effect noted above, neuropeptides must be added at the start of the culture. IFN-alpha and IFN-gamma were found to inhibit both IgE and IgG4 production whereas prostaglandin E2 (PGE2) inhibited only IgE production. However, the inhibition of IgE and IgG4 production by neuropeptides could not have been mediated by IFN-alpha, IFN-gamma, or PGE2 because the addition of anti-IFN-alpha, anti-IFN-gamma, and indomethacin, respectively, did not reverse the inhibition. In contrast to their effects on MNC, neuropeptides did not affect production of either IgE or IgG4 by purified B cells; the addition of either T cells or monocytes to B cells had no effect on this. However, neuropeptides were effective in inhibiting IgE and IgG4 production by B cells cultured together with both T cells and monocytes. Depletion of sIgE+ and sIgG4+ B cells resulted in abrogation of IgE and IgG4 production, respectively. However, stimulation of sIgE- B cells with IL-4 plus anti-CD 40 mAb induced IgE production, which was inhibited by VIP and SOM, but not SP, in the presence of both T cells and monocytes. These results suggest that neuropeptides inhibited spontaneous IgE and IgG4 production by interaction with sIgE+ and sIgG4+ B cells in a T cell- and monocyte-dependent fashion. In addition, VIP and SOM also inhibited IgE production by modulating switching induced by IL-4 plus anti-CD 40 mAb in a T cell- and monocyte-dependent fashion.
Analysis of cytokine production by Mycobacterium-reactive T cells. Failure to explain Mycobacterium leprae-specific nonresponsiveness of peripheral blood T cells from lepromatous leprosy patients.
Authors: Mutis T , Kraakman E M , Cornelisse Y E , Haanen J B , Spits H , De Vries R R , Ottenhoff T H
Published: December 2022

Recent analyses of antimycobacterial T cells clones from a small number of individuals indicate that mycobacteria preferentially induce Th cells that produce high levels of IFN-gamma and no or little IL-4 in Mycobacterium leprae-resistant tuberculoid leprosy (TT) patients and healthy subjects, whereas in one study M. leprae-induced Ts clones from polar lepromatous leprosy (LL) patients showed a reciprocal cytokine secretion profile and mediated their suppressive activity via the release of high levels of IL-4. We have evaluated these findings in peripheral blood T cells from a larger panel of TT and LL patients as well as healthy individuals. Mycobacterium-reactive T cell lines generated from the PBMC of these individuals were tested for cytokine secretion and proliferative capacity in response to M. leprae, Mycobacterium tuberculosis, and various individual mycobacterial Ag. The lepromatous pole of the leprosy spectrum was additionally investigated by analyzing the cytokine-secretion profile of M. leprae-induced (suppressor) T cell clones as well as primary ex vivo PBMC. All T cell lines from healthy individuals and TT patients responding to M. leprae, M. tuberculosis, or individual Ag, produced high levels of IFN-gamma and TNF-alpha but little or no IL-4 and IL-6. At the lepromatous pole, T cell lines failed to proliferate upon stimulation with M. leprae but in some cases produced significant levels of IFN-gamma. No IL-4 or IL-6 secretion was observed in response to M. leprae. These lines displayed strong proliferation and Th1-like cytokine production upon stimulation with M. tuberculosis. Similarly, stimulation of primary PBMC from LL patients with M. leprae or M. tuberculosis resulted in the release of IFN-gamma but no detectable IL-4 production. Control tetanus toxoid-reactive T cell lines from the same individuals instead produced large amounts of IL-4 and low levels of IFN-gamma. The analysis of M. leprae-induced T cell clones, including those with known suppressive activity, revealed that all lepromatous T cell clones produced large amounts of IFN-gamma. Most of these clones released no or little IL-4, but some clones produced higher levels of IL-4 in addition to IFN-gamma. Most clones tested produced IL-10 as well. The suppressor activity of suppressor T cell clones could not be inhibited by a neutralizing anti-IL-4 antibody and only in one case by neutralizing anti-IL-10 antibody. Anti-IL-4 and anti-IL-10 could not overcome the M. leprae-specific unresponsiveness observed in primary PBMC from LL patients.(ABSTRACT TRUNCATED AT 400 WORDS)
A synthetic peptide analogue of a determinant of type II collagen prevents the onset of collagen-induced arthritis.
Authors: Myers L K , Rosloniec E F , Seyer J M , Stuart J M , Kang A H
Published: December 2022

The immunization of genetically susceptible strains of mice with type II collagen (CII) elicits a collagen-induced arthritis that resembles rheumatoid arthritis. Our laboratory previously identified a region of CII, CII-245-270, that contains a T cell epitope that is prominent in the immune response to CII. Residues critical to the I-Aq-restricted presentation of this determinant have been characterized. To produce synthetic peptides with the potential of disrupting I-Aq-restricted Ag presentation, synthetic analogue peptides were developed that contain site-directed substitutions in critical positions. One analogue peptide was found to be an efficient competitor for binding to I-Aq and to be capable of inhibiting T cell responses in vitro. When DBA/1 mice were coimmunized with CII and the analogue peptide, the incidence and severity of arthritis were greatly reduced, concordant with the humoral immune responses to CII.
An HIV-1 envelope protein vaccine elicits a functionally complex human CD4+ T cell response that includes cytolytic T lymphocytes.
Authors: Stanhope P E , Liu A Y , Pavlat W , Pitha P M , Clements M L , Siliciano R F
Published: December 2022

T cell responses play a critical role in host defense against viral infection. Therefore, the functional properties of HIV-1-specific human T cells induced by an experimental AIDS vaccine were analyzed in detail at the clonal level. Seronegative human volunteers were immunized with a purified recombinant form of the HIV-1 envelope glycoprotein gp160 in a phase I vaccine trial. In a subset of gp160 recipients, this vaccine was shown to elicit a virus-specific CTL response. Antibody blocking and single cell cloning experiments demonstrated that the vaccine-induced cytolytic activity was mediated by CD4+, MHC class II-restricted T cells. Because little is known about the regulation of CD4+ CTL in any system, a detailed analysis of CTL responses in vaccinees was carried out. Longitudinal and cross-sectional studies revealed that the CD4+ CTL response was regulated in a complex manner and was not clearly correlated with MHC class II genotype, Ag dose, or number of immunizations. Cloning studies were carried out to determine what fraction of the vaccine-induced T cells were cytolytic and to examine patterns of cytokine production by vaccine-induced T cells. These experiments demonstrated that, for some vaccinees, CD4+ CTL dominated the in vitro T cell response to gp160 at certain time points. The level of cytolytic activity, which was a stable property of individual clones, varied among clones over a wide and continuous range. Analysis of cytokine secretion by gp160-specific CD4+ T cell clones revealed Th0-, Th1-, and Th2-like patterns, with CD4+ CTL clones showing Th0- or T'1-like patterns. Interestingly, many Th0- and Th1-like CTL clones produced very little IL-2, a finding that may explain the complicated regulation of this response. These results illustrate the complex nature of the human T cell response to subunit vaccines consisting of purified recombinant viral proteins.
Surface-exposed conserved region of the streptococcal M protein induces antibodies cross-reactive with denatured forms of myosin.
Authors: Vashishtha A , Fischetti V A
Published: December 2022

Vaccines based on a highly conserved cell surface exposed C-repeat region of the group A streptococcal M protein molecule have been found to induce protection against mucosal challenge by homologous and heterologous streptococcal serotypes. Rabbit hyperimmune antisera were produced to four partially overlapping peptides of the C-repeat region of M6 protein. These were examined by both direct and competitive ELISA and by Western blotting for their reactivity against mammalian coiled coil proteins such as laminin, myosin, light meromyosin, heavy meromyosin, and cardiac tropomyosin, and to the denatured forms of some of these molecules. All sera reacted strongly with the recombinant M6 protein molecule. In addition, antibodies to three of the peptides displayed generally low levels of cross-reactivity with at least one of the mammalian proteins, whereas antibodies to one peptide did not cross-react with any of the proteins tested. The observed reactivity was found to be directed predominantly against denatured forms of the mammalian molecules. For instance, the cleaved forms of myosin bound better to the cross-reactive antibodies than the intact molecule. Furthermore, heat-denatured heavy meromyosin competed severalfold better in competitive ELISA than the non-heat-denatured "native" form. Our results demonstrate that M protein peptides corresponding to epitopes shared among rheumatic fever-associated strains of streptococci can lead to the production of low levels of antibodies reactive with mammalian coiled coil molecules. These antibodies are directed against the denatured forms of these molecules.