bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules.
Authors: Ryan J C , Turck J , Niemi E C , Yokoyama W M , Seaman W E
Published: December 2022

In mice, the NK1.1 alloantigen is expressed on all NK cells and it initiates transmembrane signals that activate cytotoxicity. NK1.1 has been mapped to a chromosomal region that encodes two families of receptor-like molecules, the NKR-P1 family and the Ly-49 family. Both of these gene families encode type II integral membrane proteins whose extracellular domains are homologous with known C-type lectins. We have isolated and expressed a member of the NKR-P1 gene family (mNKR-P1.9) and have demonstrated both by immunofluorescence and by immunoprecipitation that this cDNA encodes NK1.1. These findings, which demonstrate the receptor-like structure of NK1.1, will facilitate studies regarding the role of NK1.1 in natural killing and regarding the identification of possible NK1.1 ligands.
Pattern of IL-1 receptor gene expression suggests role in noninflammatory processes.
Authors: Deyerle K L , Sims J E , Dower S K , Bothwell M A
Published: December 2022

The cytokine IL-1 can be elaborated by most nucleated cell types and exerts its pleiotropic effects on a variety of tissues through binding to its cognate receptor. Two forms of IL-1R, designated types I and II, have been identified. These proteins share limited amino acid identity but both bind IL-1 alpha and IL-1 beta. A large number of cell types have been shown to possess IL-1R in vitro, but few studies have addressed the question of in vivo expression. Using in situ hybridization in normal adult BALB/c mice, we have examined the tissue distributions of both IL-1R types. The results demonstrate that although lymphoid tissues in healthy animals express low or nondetectable levels of receptor transcript, nonlymphoid tissues constitutively express readily detectable levels of IL-1R mRNA. Thymus and spleen samples were largely negative for both IL-1R types I and II, whereas half of the lymph nodes examined expressed IL-1R type I. In nonlymphoid tissues, IL-1R type I transcript was detected in the dentate gyrus of the brain, endocrine pancreas, cardiac endothelium, epidermis, dermis, hair follicle epithelium, uterine serosa, vaginal stroma, vaginal squamous epithelium, developing oocytes, and granulosa cells of ruptured follicles. In contrast, the IL-1R type II probe hybridized to epidermis, dermis, and vaginal basal epithelium. The two types of IL-1R were rarely, if ever, co-expressed by the same cell. The distributional segregation of the receptor types and the expression of IL-1R in normal nonlymphoid tissues has broad implications for the role of IL-1 in homeostatic physiology.
Fetal skin: a site of dendritic epidermal T cell development.
Authors: Elbe A , Kilgus O , Strohal R , Payer E , Schreiber S , Stingl G
Published: December 2022

Thy-1 Ag and CD3-associated TCR-gamma (V gamma 3)/delta (V delta 1) are coexpressed on virtually all dendritic epidermal T cells (DETC) in the adult mouse. In contrast, day 16 fetal mouse skin contains small numbers of CD45+/Thy-1+/CD3- but no CD3+ cells. To see whether the CD45+/Thy-1+/CD3- fetal skin cells can qualify as DETC precursors, we transplanted day 16 fetal skin of C57BL/6 (Thy-1.2) mice onto adult B6Pl-Thy-1a (Thy-1.1) animals. At certain time points after transplantation, grafts were analyzed for the presence of Thy-1 and CD3/TCR Ag. Examination of the grafts, 4 days after transplantation, revealed the presence of few donor-type Thy-1.2+/CD3- and some Thy-1.2+/CD3+ epidermal cells of either round or dendritic configuration. At 10 weeks after transplantation, essentially all CD45+/Thy-1.2+ epidermal cells were anti-TCR V gamma 3 and anti-CD3-reactive, displayed a uniformly dendritic configuration, and, thus, represent DETC. Our assumption that CD45+/Thy-1+/CD3- cells are the only lymphocytes within day 16 fetal skin gained additional support by the observations: 1) that unfractionated as well as anti-CD45 gated single cell suspensions prepared from day 16 fetal skin were consistently devoid of anti-CD3 epsilon and anti-TCR V gamma 3-reactive cells; and 2) that stimulation of these cell suspensions with either Con A plus IL-2 or IL-2 alone regularly resulted in the outgrowth of CD45+/Thy-1+/CD3-/TCR V gamma 3- cells, but never in the appearance of CD45+/Thy-1+/CD3+/TCR V gamma 3+ cells. Our additional finding that Con A plus IL-2- or IL-2-stimulated day 16 fetal skin cells and cell lines derived therefrom contain transcripts of some (CD3 gamma, TCR C beta, TCR C gamma 1, TCR C gamma 4) but not of other (CD3 delta, CD3 epsilon, TCR C alpha, TCR C delta) genes encoding the CD3/TCR complex suggests that Thy-1+/CD3- fetal murine skin cells are of T cell lineage. We therefore propose that the fetal skin microenvironment can provide the stimuli promoting growth and maturation of CD3/TCR V gamma 3/V delta 1-expressing DETC from their CD3- precursors.
Soluble complement receptor type 1 ameliorates the local and remote organ injury after intestinal ischemia-reperfusion in the rat.
Authors: Hill J , Lindsay T F , Ortiz F , Yeh C G , Hechtman H B , Moore F D
Published: December 2022

We examined the role of C activation in ischemia reperfusion injury by inhibiting C activation in a rat model of mesenteric arterial occlusion. In anesthetized rats, 60 min of mesenteric arterial occlusion was followed by 3 h of reperfusion. PBS alone or containing soluble C receptor 1 (3 or 6 mg) was administered i.v. Controls underwent laparotomy without ischemia. Relative serum C activities were assessed by hemolytic assay, neutrophil (polymorphonuclear leukocyte) sequestration by tissue content of myeloperoxidase (MPO) activity, intestinal mucosal injury by histologic grading, lung vascular permeability by the ratio of bronchoalveolar lavage to blood concentration of radiolabeled BSA, and endothelial cell injury was quantified by measurement of plasma factor VIII-related Ag. After reperfusion, PBS-treated animals had increased intestinal MPO (0.048 +/- 0.007 U/g) compared to sham (0.022 +/- 0.005 U/g (p less than 0.05)) and intestinal mucosal injury score (2.490 +/- 0.221) compared to sham (0.331 +/- 0.045 (p less than 0.05)). Treatment with 6 mg soluble C receptor 1 15 min before reperfusion reduced intestinal MPO (0.017 +/- 0.003 U/g (p less than 0.05)) and mucosal injury (1.733 +/- 0.168 (p less than 0.05)) compared to PBS control. PBS-treated animals also demonstrated increased lung MPO (0.314 +/- 0.025 U/g vs 0.085 +/- 0.018 in sham (p less than 0.05)) and increased lung permeability (bronchoalveolar lavage/blood cpm 11.32 +/- 1.35 x 10(-3) vs sham 2.22 +/- 0.19 x 10(-3) (p less than 0.05)). Treatment with 6 mg soluble C receptor 1 15 min before reperfusion or at reperfusion reduced the lung permeability (bronchoalveolar lavage/blood cpm 3.90 +/- 0.79 x 10(-3) and 5.08 +/- 0.75, respectively (both p less than 0.05)) compared to PBS control, but did not reduce lung MPO (0.342 +/- 0.031 U/g and 0.246 +/- 0.025), respectively. Treatment with sCR1 also reduced the release of factor VIII-related Ag, 5-day mortality, and C hemolytic activity. In this model, C is a major mediator of intestinal injury and extraintestinal injury.
Tissue distribution of human minor histocompatibility antigens. Ubiquitous versus restricted tissue distribution indicates heterogeneity among human cytotoxic T lymphocyte-defined non-MHC antigens.
Authors: de Bueger M , Bakker A , Van Rood J J , Van der Woude F , Goulmy E
Published: December 2022

We determined the tissue distribution of 7 human minor histocompatibility (H) Ag. Each of these Ag is defined by one or more MHC class I-restricted CTL clones, previously generated from PBL primed against minor H Ag by HLA-identical bone marrow transplantation (BMT). CTL-mediated lysis of tissue-derived cells and cultured cell lines was used as an in vitro assay for minor H Ag expression of several human tissues. The Ag HA-3 (HLA-A1-restricted), HA-4 (HLA-A2 restricted), HA-6 and HA-7 (HLA-B7 restricted), and the male-specific Ag H-Y (HLA-A2 and B7 restricted) were found to be expressed on cells of all tissues tested. In contrast, the HLA-A2-restricted Ag HA-1 and HA-2 were demonstrated on PHA-blasts, EBV-BLCL, purified T cells, B cells, monocytes, and immature thymocytes, but could not be demonstrated on skin-derived cultured fibroblasts, keratinocytes, melanocytes, cultured epithelial cells of kidney proximal tubili, and umbilical cord vein-derived endothelial cells. Incubation of the latter cell lines with rIFN-gamma, rTNF-alpha, and/or rIL-1 alpha, in concentrations shown to maximally increase their susceptibility to lysis by allo-MHC class I CTL, did not induce recognition by HA-1- and HA-2-specific CTL in vitro. These results indicate an ubiquitous tissue expression of the minor H Ag HA-3, -4, -6, -7 and H-Y in contrast to a to the hemopoietic cell lineage-restricted expression for HA-1 and HA-2. The heterogeneity in tissue expression of T cell-defined, class I-restricted non-MHC Ag implies that they might be derived from intracellular proteins with either an ubiquitous or a more specialized cell type-specific function.
Heteroclitic polyclonal and monoclonal anti-Gm(a) and anti-Gm(g) human rheumatoid factors react with epitopes induced in Gm(a-), Gm(g-) IgG by interaction with antigen or by nonspecific aggregation. A possible mechanism for the in vivo generation of rheumatoid factors.
Authors: Williams R C , Malone C C , Casali P
Published: December 2022

Heteroclitic rheumatoid factors (RF) are specific for allotypic determinants, e.g., Gm(a) or Gm(g) on allogeneic, but not autologous IgG. All polyclonal RF we isolated from nine rheumatoid arthritis patients with circulating Gm(a-), (b+), (g-), (f+) IgG displayed dual heteroclitic activity for the Gm(a) and Gm(g) allotypes, as shown by using appropriate RBC agglutination assays and affinity columns bearing Gm(a+) or Gm(g+) IgG. To investigate possible mechanisms underlying the in vivo generation of heteroclitic RF, we tested the ability of nonspecifically and immune-specifically aggregated Gm(a-), (g-) IgG to function as targets for RF from Gm(a-), (g-) patients with rheumatoid arthritis. Heat aggregation (63 degrees C for 20 min) or binding to Ag (as in tetanus toxoid-antitetanus toxoid complexes) induced a "functional" Gm(a+) and/or (g+) phenotype in Gm(a-), (g-) IgG from five healthy subjects and five rheumatoid patients, as suggested by the ability of these altered IgG to function as efficient targets for six heteroclitic RF in direct binding and competitive inhibition experiments. That heterocliticity and dual Gm(a), Gm(g) specificity can be features of a single antibody molecule was formally demonstrated by analysis of a monoclonal RF (IgM mAb 61) generated from a Gm(a-), (g-) rheumatoid patient. RF mAb 61 displayed a high affinity (Kd, 10(-7) M) for IgG Fc fragment of Gm(a+) and (g+) IgG or aggregated autologous Gm(a-), (g-) IgG but did not bind to native autologous IgG. To investigate the molecular basis of the acquired Gm(a) phenotype, PBMC from five Gm(a-) patients with rheumatoid arthritis and two Gm(a-) normal subjects arthritis and two Gm(a-) normal subjects were cultured in vitro after activation with PWM. In most instances, these PBMC produced IgG that behaved as Gm(a+) in sensitive ELISA. Application of the polymerase chain reaction (PCR), using probes specific for the nucleotide sequence coding for the Gm(a) tetrapeptide, to the amplification of DNA from the in vitro-stimulated Gm(a-) normals or rheumatoid patients' PBMC provided no evidence for Gm(a) nucleotide sequences. The present data suggest that acquisition of the Gm(a) determinant by Gm(a-) IgG may result from subtle changes in the CH2-CH3 RF-binding region. Such changes would occur when Gm(a) IgG are complexed with Ag or nonspecifically altered, thereby providing a possible explanation for the induction of heteroclitic RF in Gm(a-) rheumatoid arthritis patients.
Prolonged IL-2 receptor alpha/CD25 expression after T cell activation via the adhesion molecules CD2 and CD28. Demonstration of combined transcriptional and post-transcriptional regulation.
Authors: Cerdan C , Martin Y , Courcoul M , Brailly H , Mawas C , Birg F , Olive D
Published: December 2022

The T cell adhesion molecule CD28 provides a costimulatory signal in combination with CD2 and CD3 mAb. CD28 regulates the expression of cytokines by T cells, not only IL-2, but also IL-1 alpha and CSF-1, usually synthesized by accessory cells. We have investigated the mechanisms through which CD28 modulates the expression of the IL-2R alpha chain. Whereas activation through CD2 or CD28 alone induced no or only low IL-2R alpha chain expression, activation through CD2 plus CD28 led to both a high and prolonged (greater than 14 days) cell surface and mRNA expression. In contrast, immobilized CD3 mAb-dependent activation induced a transient expression of the IL-2R alpha chain, which was neither further increased nor prolonged by CD28 costimulation. Upon CD2 plus CD28 stimulation, the half-lives of the two IL-2R alpha transcripts increased progressively between days 1 and 4, in contrast to each pathway alone. Whereas each activation pathway alone induced either no (CD2) or low (CD28) levels of IL-2R alpha gene transcription, the CD2 plus CD28 stimulation was associated with its increased transcription, which persisted at similar rates between 5 and 96 h post stimulation. The in vitro costimulation via the CD2 and CD28 molecules thus regulates the expression of the IL-2R alpha gene both at the transcriptional and post transcriptional levels. Our results therefore demonstrate a new immunoregulatory function of the CD28 molecule on IL-2R alpha expression, which, through its increased transcription rate and stabilization, could, together with high levels of cytokines secretion, be responsible for the prolonged T cell proliferation.
Activation requirements for CD4+ T cells differing in CD45R expression.
Authors: Luqman M , Bottomly K
Published: December 2022

Murine CD4+ T cells can be subdivided into naive and memory T cells based on surface phenotype, on recall response to Ag, and on differences in activation requirements. Furthermore, several studies have shown that two signals are required for CD4+ T cell activation; one signal is provided by occupancy of the TCR and the other signal is provided by the APC. In this report, analysis of naive and memory CD4 T cells, separated on the basis of CD45 isoform expression, has shown that their requirements for two signals differ. Activation of memory CD4 T cells to proliferate and secrete IL-2/IL-4 only required occupancy of the TCR complex, whereas activation of naive CD4 T cells required an APC-derived signal as well. Moreover, the signal induced by anti-CD3 antibodies differs from the signal provided by anti-V beta cross-linking of the TCR because both antibodies activate memory CD4 T cells but only anti-CD3 activates naive CD4 T cells. Together these data suggest that the consequence of stimulation through the TCR/CD3 signal complex differs between memory and naive CD4 T cells.
Lipopolysaccharide induces DNA binding activity specific for the IFN-stimulated response element in murine peritoneal macrophages.
Authors: Tebo J M , Hamilton T A
Published: December 2022

Several murine macrophage genes that exhibit transcriptional response to LPS (e.g., IP-10, D3) have IFN-stimulated response element (ISRE) sequences present in regions flanking the transcription start sites. In the present study, the ability of LPS to activate proteins in murine peritoneal macrophages capable of binding to the ISRE has been investigated. Nuclear extracts from both LPS-treated and untreated macrophages are capable of forming four distinct complexes with a radiolabeled oligonucleotide containing the ISRE sequence as detected by electrophoretic mobility shift assays. LPS-treated nuclei contained an additional ISRE binding activity (complex I) that was not found in unstimulated cells. The induction of the latter activity by LPS was sensitive to polymyxin B sulfate, a lipid A antagonist, demonstrating that LPS was the primary inducing activity. All five retarded protein-DNA complexes formed were specific for the ISRE sequence as shown by competition with a series of oligonucleotides containing either ISRE-related or -unrelated sequences. Complex I binding activity was dependent upon the concentration of LPS and the time of LPS treatment. Furthermore, complex I was similar to that induced in response to treatment with IFN-beta in terms of electrophoretic mobility and specificity for the ISRE. LPS-induced complex I formation was partially independent of protein synthesis and could not be blocked by including neutralizing antibody to IFN-alpha/beta in the culture medium. Thus, even though LPS is a potent inducer of IFN-beta in murine macrophages, class I IFN expression may not be an obligatory intermediate event in the LPS-driven activation of ISRE binding activity. These results suggest that induction of ISRE binding activity may be an important part of the signaling process initiated by LPS.
Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha.
Authors: Aloisi F , Carè A , Borsellino G , Gallo P , Rosa S , Bassani A , Cabibbo A , Testa U , Levi G , Peschle C
Published: December 2022

Astrocyte-enriched populations were established from human embryonic brain analyzed for their ability to synthesize cytokines potentially relevant for mechanisms of inflammation and immunity in the brain. Unstimulated astrocytes did not secrete significant IL-6, IL-8, macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), or granulocyte-CSF (G-CSF), as determined by specific ELISA and/or bioassay. With the exception of M-CSF mRNA, transcripts for the above factors were not detected in unstimulated astrocytes. On exposure of human astrocytes to IL-1 beta, high levels of IL-6, IL-8, M-CSF, G-CSF, and GM-CSF mRNAs were detected; moreover, active secretion of all the above cytokines was demonstrated. TNF-alpha was also able to stimulate IL-6, IL-8, M-CSF, GM-CSF, and G-CSF synthesis and secretion, but was generally less potent than IL-1 beta. No IL-3 mRNA or protein was detected in unstimulated or cytokine-treated astrocytes. IL-1 alpha and IL-1 beta mRNAs and proteins were not detected in unstimulated astrocytes, but were present in very small amounts after stimulation with TNF-alpha/IL-1 beta. No IL-6, M-CSF, GM-CSF, G-CSF, or IL-8 were induced by IL-1 beta or TNF-alpha in early primary cultures, which mainly contain undifferentiated neuronal/glial progenitor cells. These studies demonstrate for the first time the production of multiple cytokines by normal human astrocytes stimulated in culture by IL-1 beta and TNF-alpha. The capacity of human astrocytes to synthesize and release cytokines active on hemolymphopoietic cells supports the concept that these cells play an important role in the regulation of inflammatory and immune responses in a variety of brain pathologies.
Protein kinase C isoenzymes display differential affinity for phorbol esters. Analysis of phorbol ester receptors in B cell differentiation.
Authors: Marquez C , Martinez C , Kroemer G , Bosca L
Published: December 2022

Protein kinase C (PKC) comprises a family of distinct isoenzymes that are involved in signal transduction pathways linking the cell to triggers perceived via membrane receptors. These isoenzymes differ in their tissue distribution, activation requirements, and substrate specificity. One common denominator among different PKC subspecies is their activation by phorbol esters. We have developed a sensitive method permitting the measurement of phorbol ester binding sites, their quantitation, as well as their dissociation kinetics, by performing cytofluorometric analyses on intact cells or on isolated PKC associated to phosphatidylserine vesicles incubated in the presence of fluorochrome-labeled phorbol ester. Both PKC isozymes beta I/beta II and alpha from brain and spleen after incorporation into phosphatidylserine vesicles, display affinities with apparent Kd of 120 and 50 nM, respectively; although PKC gamma from brain exhibits a Kd of 210 nM. In addition to these receptors, on PKC isozymes from spleen, an intermediate affinity phorbol ester receptor (Kd of 3 nM) and an additional high affinity phorbol ester binding site with a Kd of 0.1 to 0.5 nM were also detected. This latter receptor comigrates with high m.w. PKC isoforms. In different cell lines, the phorbol ester binding patterns, as well as the expression of individual PKC isoenzymes, could be positively correlated.
Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.
Authors: Gerard C , Bao L , Orozco O , Pearson M , Kunz D , Gerard N P
Published: December 2022

The mouse C5a receptor gene was isolated using the human C5a receptor cDNA probe recently described (Gerard, N. P., and C. Gerard. 1991. Nature 349:614). By analogy with the human gene, the mouse homolog contains two exons with the 5' untranslated region and initiating methionine codon present in exon 1 and the remainder of the molecule in exon 2. Generation of an expressible cDNA for the mouse C5a receptor was accomplished using the polymerase chain reaction and a sense oligodeoxynucleotide primer which included an initiation codon just 5' to the sequence encoding the N-linked glycosylation site. When transfected into human 293 kidney epithelial cells the cloned cDNA directs expression of a binding site for human C5a anaphylatoxin with a binding constant of 2.5 +/- 0.3 nM; the human C5a receptor expressed under identical conditions has a Kd of 1.7 +/- 0.2 nM. Overall, the deduced amino acid sequences of the receptors are 65% identical given the analogous gene structures. Alignment of the sequences as seven transmembrane segment receptors reveals that the greatest structural diversity (approximately 70%) exists in the putative extracellular domains. In contrast, species differences among other members of this family of seven membrane-spanning receptors is generally only 10 to 20%, even for receptors whose ligands are relatively small and not expected to interact with sites on the extracellular surfaces. A high degree of structural identify is observed for the C5a receptors in the transmembrane segments and in all but one of the loops predicted to exist in the cytoplasm. Inasmuch as critical structures responsible for high affinity binding of the 74 amino acid polypeptide to both C5a receptors involve features conserved between species, these data provide the starting point for mutagenesis studies to determine the nature of the binding and activation sites for the chemotactic receptors. Additionally, these data provide a reagent for immunologic and molecular genetic studies on the role of C5a receptors in inflammatory models.
Autocrine growth limitation of human papillomavirus type 16-harboring keratinocytes by constitutively released tumor necrosis factor-alpha.
Authors: Malejczyk J , Malejczyk M , Köck A , Urbanski A , Majewski S , Hunzelmann N , Jablonska S , Orth G , Luger T A
Published: December 2022

TNF-alpha is known to exert antitumor and antiviral effects and to participate in the regulation of the immune response. In our study we demonstrate that human rTNF-alpha specifically blocks growth of SK-v keratinocyte cell line harboring and expressing human papillomavirus type 16 (HPV16) sequences. This inhibitory effect was shown by [3H]TdR incorporation and cell counting. Binding experiments with 125I-TNF-alpha showed that SK-v cells express about 10,000 single class TNF-alpha R per cell with affinity constant of about 0.7 nM. Binding of 125I-TNF-alpha could be inhibited by htr-9 mAb recognizing a 55/60-kDa type I TNF-alpha R but not by utr-1 mAb recognizing 75/80-kDa type II TNF-alpha R or irrelevant mAb specific for HPV16E7 protein. Addition of anti-TNF-alpha antibodies to SK-v cell culture resulted in significant (p < 0.05), dose-dependent stimulation of their proliferation. SK-v cells constitutively expressed TNF-alpha mRNA, and SK-v CM contained TNF-alpha, as demonstrated by Northern blot analysis, a specific ELISA, Western blot analysis, and a bioassay with TNF-alpha-sensitive L-M cells. HPLC gel filtration of SK-v cell CM showed that the factor cytotoxic for L-M cells coeluted with immunoreactive TNF-alpha. These results demonstrate that HPV16-harboring SK-v cells constitutively express and release immunoreactive and biologically active TNF-alpha that in turn may exert an autocrine growth inhibitory effect. This phenomenon could represent one of the self-limiting mechanisms controling growth of HPV-induced neoplasia.
Changes in the precursor frequencies of IL-4 and IFN-gamma secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis.
Authors: Morris L , Troutt A B , Handman E , Kelso A
Published: December 2022

Limiting dilution analysis was used to estimate the frequency of clonogenic Ag-specific CD4+ T lymphocytes in draining lymph nodes of mice over the course of infection with Leishmania major, and to measure the production of IL-2, IL-3, IL-4, IFN-gamma, and TNF by the resultant clones. Infection of both genetically susceptible BALB/c ("non-healer") and resistant C57BL/6 ("healer") mice resulted in at least a fourfold increase in the frequency (to about 0.3%) and at least a 10-fold increase in the total number of lymph node CD4+ cells that formed clones when cultured with L. major Ag in vitro. At 1 wk after infection, the majority of clones from BALB/c mice secreted IL-4 (precursor frequency 0.15%) and fewer secreted IFN-gamma (0.05%); this pattern remained constant for at least 8 wk after infection. In C57BL/6 mice, however, a high precursor frequency of IL-4-secreting clones was measured in the first 1 to 2 wk when the mice had lesions, but resolution of infection was associated with a decrease in the frequency of IL-4-secreting clones (from 0.13% at 2 wk to 0.03% at 4 wk) and an increase in the frequency of IFN-gamma-secreting clones (from 0.08% to 0.22%). At all stages of infection, most clones from either mouse strain secreted IL-3 and very few secreted TNF. Analysis of PCR-amplified cDNA from draining lymph nodes of infected mice also revealed that IL-4 and IFN-gamma mRNA were expressed in both mouse strains early in infection. IL-4 mRNA was the major species at 2 and 6 wk after infection in BALB/c mice, but declined relative to IFN-gamma mRNA over this time in C57BL/6 lymph nodes. Precursor frequency estimates of lymphokine-secreting CD4+ cells in draining lymph nodes therefore correlated with lymphokine expression patterns in vivo. Analysis of a panel of individual short term clones derived from mice 1 wk after infection revealed marked heterogeneity in lymphokine production patterns. In BALB/c mice, 49% secreted IL-4 without IFN-gamma, 18% secreted IFN-gamma without IL-4, and 14% secreted both IL-4 and IFN-gamma. Similarly in C57BL/6 mice, 39% secreted IL-4, 20% secreted IFN-gamma, and 17% secreted both lymphokines. Many of the clones also produced IL-3 and/or IL-2. Together the data suggest that both IL-4 and IFN-gamma are synthesized early in infection of susceptible and resistant mice as assessed by mRNA and precursor frequency analyses.(ABSTRACT TRUNCATED AT 400 WORDS)
Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.
Authors: Kreitman R J , Schneider W P , Queen C , Tsudo M , Fitzgerald D J , Waldmann T A , Pastan I
Published: December 2022

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.
T cell differentiation antigens on lymphocytes in the human intestinal lamina propria.
Authors: Schieferdecker H L , Ullrich R , Hirseland H , Zeitz M
Published: December 2022

Recently, T cell subpopulations presumably representing memory T lymphocytes have been described in vitro. Intestinal lamina propria T cells (LP-T) have characteristics resembling those of memory cells. We therefore investigated the expression of surface Ag associated with memory phenotype in vitro on lamina propria lymphocytes (LPL) and PBL and on the T cell subpopulations defined by the bright expression of CD45R0 by flow cytometric analysis of isolated cell populations. LPL had significantly increased percentages of CD45R0 and CD58 positive cells compared with PBL. Whereas PBL showed bimodal expression profiles of CD45R0, CD58, and CD2, the vast majority of LPL was bright for these Ag. Expression of CD45RA was significantly reduced in both frequency and intensity in LPL, and LPL had significantly reduced percentages of CD11a/CD18 and CD29 positive cells compared with PBL. The CD45R0 bright T cell subpopulations of both PBL and LPL were characterized by a lack of CD45RA. CD45R0 bright T cells from the peripheral blood (PB-T) were predominantly bright for CD2, CD58, CD29, and CD11a/CD18 whereas CD45R0 dim PB-T had bimodal expression profiles and CD45R0 negative PB-T were dim or even negative for these Ag. CD45R0 bright LP-T were also bright for CD2 and CD58 but had significantly reduced surface densities of CD11a/CD18 and CD29 compared with CD45R0 bright PB-T. The surface density of CD29 on CD45R0 bright LP-T corresponded to that of CD45R0 negative PB-T, and a significant proportion of CD45R0 bright LP-T was even negative for CD11a/CD18 and CD29. Additionally, CD45R0 bright LP-T in contrast to PB-T were characterized by a lack of 1-selectin and the expression of CDw49a and the mucosa-specific T cell Ag HML-1 on high percentages of cells. Our results show that the phenotype of CD45R0 bright T cells from the lamina propria clearly deviates from that of memory T cells in vitro and of CD45R0 bright T cells in the peripheral blood. We conclude that memory T cell populations in vivo undergo specific differentiation depending on their tissue localization, leading to unique phenotypic and presumably functional features.
Anergic Th1 cells express altered levels of the protein tyrosine kinases p56lck and p59fyn.
Authors: Quill H , Riley M P , Cho E A , Casnellie J E , Reed J C , Torigoe T
Published: December 2022

Tolerance in T lymphocytes can result from clonal anergy, or paralysis, of Ag-specific T cells. To investigate the molecular mechanisms responsible for anergy, a system in which tolerance can be induced in vitro was employed. Anergy, as defined by long-lived nonresponsiveness to normal antigenic stimulation for IL-2 production, was produced in cloned murine CD4+ Th1 cells. Here we report that such anergic Th1 cells express constitutively reduced amounts of the protein tyrosine kinase p56lck and constitutively elevated levels of the protein tyrosine kinase p59fyn. Because protein tyrosine phosphorylation is known to be important for the normal induction of IL-2 synthesis, these results suggest that T cell anergy may be maintained, at least in part, by alterations in tyrosine phosphorylation signaling events.
Restriction fragment length polymorphism of the murine C4 and Slp genes: two C4 groups.
Authors: Miller R D , Aizawa Y , Shreffler D C
Published: December 2022

We have examined the related H-2 genes coding for the fourth component of complement (C4) and the sex-limited protein (Slp) from 30 inbred mouse strains by Southern blot analysis. With four restriction enzymes, 11 RFLP patterns distributed among 26 different H-2 haplotypes have been identified. Strains of the same serologic H-2 haplotype were found to have identical RFLP patterns. It was confirmed that the number of C4-related genes in most haplotypes is two, Slp and C4; but H-2SWI6 (SWI6) and SWI9, which have the same RFLP pattern, have four and Sw7 has five. Although C4 and Slp have many similarities, they also were found to contain distinctive features: relative to Slp, each C4 allele examined has two insertions totaling 1.1 kb located in introns 14 and 15; and each Slp allele examined, excluding hybrids, has a provirus insertion upstream. No other large deletions or insertions were detected. The RFLP patterns are also due to 10 polymorphic restriction sites, which have been placed on standard maps; two are associated with Slp and eight are associated with C4.Sk strains, the only strains that express low serum levels of C4, have the same RFLP phenotype as Sw14, Sw18, and Swx; Sk may have arisen from a recent common ancestor of these strains. Homologous recombination has been important in the formation of existing C4 alleles. However, based on complete linkage disequilibrium between three RFLP internal to C4, the haplotypes have been divided into two groups that may have functional significance.
Immunological Studies in Human Parasitic Infestation
Authors: Brunner Matthew
Published: December 2022

Intradermal tests with saline extracts of Ascaris and Dibothriocephalus were made upon 277 individuals of from one to sixty-five years of age, and with extracts of Taenia solium upon 20 persons. Reactions similar to those elicited in conditions of atopic hypersensitiveness (hay fever and asthma) were obtained. Of 110 moderate and slight reactions 6 (or about 6 per cent) showed infestation in the excreta, as corroborated by findings. Of 53 individuals presenting marked reactions, 21 (or 40 per cent) revealed positive findings in the stools. Of 309 cases giving negative reactions 1 was found to harbor Dibothriocephalus. The absence of a positive reaction is not an absolute criterion of the absence of infestation with Dibothriocephalus, for one person infested with Dibothriocephalus remained negative to repeated tests. Among those reacting to the Ascaris antigen were some individuals that were infested not with Ascaris lumbricoides, but with other members of the nematode family,—namely Uncinaria (hookworm), Trichiuris trichiuria, Oxyuris vermicularis, and Trichinella spiralis. This indicates a group reaction. Testing with the extract of Ascaris lumbricoides is sufficient therefore for the diagnosis of any nematode infestation. The specificity of Dibothriocephalus latus and Taenia solium reactions was demonstrated. In every case which gave a marked positive reaction, where the transfer of sensitivity passively to the normal human skin was undertaken, the transfer was successful even in individuals in whom active infestation could not be demonstrated, suggesting similarity to the same phenomenon observed in some atopic cases. Constitutional reactions elicited in 3 asthmatic individuals as the result of intradermal tests with the Ascaris extract, indicates that extreme caution is necessary for routine testing, especially in atopic persons, with this type of extract. A marked reaction was interpreted to indicate either the presence of parasites or recent infestation.
On the Anti-Hemolytic Action of Hemolytic Immune Sera
Authors: Yanagihashi Mototoshi
Published: December 2022

I. Introduction. Since Ehrlich (1) in 1898 discovered the inhibition of the hemolytic action of tetanus-toxin by normal horse serum, many investigations of anti-hemolytic action have been made.

Kraus and Clairmont (2) and Eisler (3) found that normal horse serum could inhibit the hemolytic action not only of tetanus toxin, but of staphylococci, streptococci, vibrios, and putrefactive bacilli.

Ranson (4) examining the hemolytic action of saponin against dog's blood corpuscles, discovered that the addition of dog's serum to saponin would inhibit its hemolytic action.

Human serum also has an anti-hemolytic action. But it was due to the discovery of Neisser and Doering (5), Neisser and Friedemann (6), and Senator (7) that the fact that such action of serum was possible only when it was inactivated by being heated to 56°C. became known.

Much (8) reported that the serum of subjects of psychosis has the power of inhibiting the hemolytic action of cobra toxin against human blood corpuscles.
Effect of Serological Systems on Paramecium
Authors: Rakieten Morris L.
Published: December 2022

Within the past two decades a number of investigators have studied the behavior of paramecia in artificially created environments, and many important biological phenomena have been disclosed. Most of these studies are not pertinent to the question here under consideration, but certain of them, notably those of Balbiani (1898), Ledoux-Lebard (1902), and Hamilton (1904), have a direct application, since they involve the reaction of paramecia to antibody-containing sera of various types. Balbiani and Ledoux-Lebard studied the toxic effects of various sera on paramecia, and Hamilton, working specifically with the sera of scarlet fever and pneumonia patients, demonstrated that such sera are definitely more toxic than are normal sera. Certainly the most extensive studies on the behavior of paramecia are those of Jennings (1904) and Woodruff, the first of these authors being concerned with the relation of movement, and the responses of the action system under a variety of stimuli.
Alternatively spliced pp52 mRNA in nonlymphoid stromal cells.
Authors: Gimble J M , Dorheim M A , Youkhana K , Hudson J , Nead M , Gilly M , Wood W J , Hermanson G G , Kuehl M , Wall R
Published: December 2022

The 52-kDa phosphoprotein, also reported as lymphocyte-specific gene 1 and WP34, is transcribed as a 1.6-kb mRNA in B lymphocytes, B cell lines, and untransformed T cells. This gene encodes a cytoplasmic and plasma membrane-associated protein that is phosphorylated at a casein kinase II site and reportedly binds calcium. Based on these properties, it has been hypothesized that lymphoid form of the 52-kDa phosphoprotein protein may play a role in lymphocyte signal transduction. We show that alternatively spliced mRNA are expressed from this gene in nonlymphoid cell lines (myocytes, stromal cells, fibroblasts). These cell lines do not express the 1.6-kb lymphoid cell-specific transcript. Instead, mRNA of 2.0 and 2.8 kb are detected in varying abundance. A full-length 2.0-kb cDNA has been cloned and sequenced from the BMS2 stromal cell line by conventional screening and polymerase chain reaction-based methods. This cDNA clone, designated S37, has a single open reading frame encoding a 328 amino acid peptide. The nucleotide sequence of the S37 stromal cell cDNA is identical to that of the lymphocyte derived pp52 cDNA from the 3' poly(A) tail to the codon encoding the amino acid at residue 24. This region of the S37 cDNA clone encodes a protein that is identical to that encoded by the lymphoid pp52 cDNA and includes a casein kinase II phosphorylation site. However, the two clones differ in their 5' nucleotide sequence and their NH3 terminal amino acid sequence. This organization is consistent with alternative exon utilization. These results suggest that tissue-specific control mechanisms are used to generate different forms of lymphoid form of the 52-kDa phosphoprotein mRNA in lymphoid cells versus mesoderm-derived, nonlymphoid cell lineages.
Identification of 5'-flanking regions affecting the expression of the human decay accelerating factor gene and their role in tissue-specific expression.
Authors: Thomas D J , Lublin D M
Published: December 2022

Decay accelerating factor (DAF) is a complement regulatory protein that protects host tissue from complement-mediated damage by preventing the assembly and/or promoting the dissociation of C3 and C5 convertases. To identify and analyze the DNA sequence elements responsible for controlling DAF expression, the 5'-flanking region of the human DAF gene was cloned. Sequencing of 880 nucleotides upstream from the ATG codon revealed the absence of classic TATA or CAAT boxes. RNase protection and primer extension assays revealed a series of transcription start sites in a 10 nucleotide region located 86 nucleotides upstream from the ATG (the first of these start sites is numbered +1). Using HeLa, K562, EBV, and Molt 4 cells, DAF mRNA and protein were analyzed by Northern and Western blot. The DAF mRNA and protein levels roughly correlated, suggesting transcriptional control of gene expression. K562 and HeLa expressed high levels, EBV expressed intermediate levels, and Molt 4 expressed essentially no detectible DAF mRNA or protein. Regions of DAF 5'-flanking DNA were subcloned into plasmids containing the chloramphenicol acetyltransferase reporter gene and tested in transient transfection assays. The construct extending from -206 to +84 (-206/+84) had transcriptional activity in the DAF-positive HeLa, K562, and EBV lines, but no activity in the DAF-negative Molt 4 line. In the three DAF-positive lines, major enhancer activity was demonstrated between -206 and -77, and between -77 and -54 (containing cAMP responsive element and AP-1 binding site). Additional deletion of the region between -54 and -34 (containing an Sp1 binding site) reduces chloramphenicol acetyltransferase activity further but the low numerical values preclude statistical significance. The identification of transcription start sites and enhancer regions in the DAF gene will be important for studies of the mechanisms whereby cytokines and other factors may modulate DAF expression.
Intratumor gene expression after adoptive immunotherapy in a murine tumor model. Regulation of messenger RNA levels associated with the differential expansion of tumor-infiltrating lymphocytes.
Authors: Evans R , Kamdar S J , Duffy T , Edison L
Published: December 2022

The aim of this paper was to show that the adoptive immunotherapy (AIT) of established tumors resulted in the activation of defined lymphocyte-associated genes at the site of rejection. C57BL/6J mice bearing the moderately immunogenic syngeneic MCA/76-9 sarcoma received combination therapy 10 days after tumor cell implantation. This consisted of a single i.p. injection of cyclophosphamide (CY) followed by the i.v. injection of tumor-sensitized T cells (CY/AIT). The previously observed in situ differential expansion of tumor-infiltrating lymphocytes (TIL) was associated with a parallel modulation of CD4 (L3T4) and CD8 Ag expression. Flow cytometric analysis indicated that the CD8+ TIL appeared to contain two subpopulations during the reported proliferative phase, but by day 8 after CY/AIT the cells were composed of a single bright population. The CD4+ TIL similarly appeared to show two subpopulations, but in contrast to the CD8+ TIL there was a shift to a predominantly less bright single population by day 8. The expression of lymphocyte genes (Ly-2 and Ly-4 encoding the CD8 and CD4 Ag, respectively, IL-2, IL-2R, IFN-gamma, and IL-6) was analyzed by Northern hybridization using RNA extracted from whole tumor tissue. Progressing tumors expressed only low and relatively constant levels of mRNA for all of the genes except Ly-2 over a 19-day period. In contrast, there were considerable temporal fluctuations in mRNA levels depending on whether the mice had received CY or CY/AIT, but it was apparent that CY/AIT induced the more dramatic changes as far as Ly-2, Ly-4, IL-2, and IFN-gamma mRNA levels were concerned. A preliminary survey of cytokine bioactivity released by tumor-associated cells isolated 9 days after CY/AIT indicated that both IFN-gamma and IL-6 activities were released by the cells, but not IL-2, despite the relatively high levels of IL-2 mRNA. These data provide evidence that the differential expansion of TIL after CY/AIT is accompanied by well defined changes in the levels of mRNA-encoding TIL membrane Ag and lymphokine genes and are concordant with the view that amplified anti-tumor immune responses occur after AIT.
Type 1 plasminogen activator inhibitor is not an acute phase reactant in rats. Lack of IL-6- and hepatocyte-dependent synthesis.
Authors: Podor T J , Hirsh J , Gelehrter T D , Zeheb R , Torry D , Guigoz Y , Sierra F , Gauldie J
Published: December 2022

The contributing role of hepatocytes and IL-6 in the acute phase-like elevation of plasma type 1 plasminogen activator inhibitor (PAI-1) in vivo is not known. We addressed this question by comparing the effects of two inflammatory stimuli known to increase plasma concentrations of IL-6 on the plasma concentrations and site of synthesis of PAI-1 and cysteine proteinase inhibitor (CPI) in rats. Subcutaneous injection of turpentine results in a sustained increase in plasma IL-6 and CPI Ag levels over a 24-h period. In contrast, plasma PAI-1 activity was not altered by turpentine treatment. Northern blot analysis of poly(A)+ mRNA extracted from freshly isolated hepatocytes of saline- or turpentine-treated animals demonstrated induction of CPI mRNA expression but failed to detect basal or induced PAI-1 or IL-6 mRNA expression. However, PAI-1 mRNA was detected in rat hepatocytes in primary culture for 24 h and was induced by dexamethasone. Intravenous infusion of bacterial LPS (4 mg/kg) induced a sustained increase in plasma CPI Ag and transient increases in plasma IL-6 and PAI-1. Northern blot analysis of freshly isolated, fractionated liver cells indicated that LPS treatment (3 h) induced PAI-1 mRNA expression most significantly in the endothelial and Kupffer cell fractions. IL-6 mRNA expression was induced in Kupffer cells and CPI mRNA was induced in hepatocytes. Immunocytochemical analysis revealed LPS-induced accumulation of PAI-1 Ag associated with the vascular endothelium, subendothelial matrix, and sinusoidal lining cells. Our results indicate that PAI-1 mRNA is not significantly expressed by rat hepatocytes in vivo and that plasma PAI-1 levels are not influenced by increased IL-6 expression in Kupffer cells or in plasma.