bluuebunny/crossref_metadata_2025 huggingface dataset:
Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets.
Authors: Heinzel F P , Sadick M D , Holaday B J , Coffman R L , Locksley R M
Published: June 2004

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.
A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. Differential reactivity with subsets of immature and mature T lymphocytes.
Authors: Hünig T , Wallny H J , Hartley J K , Lawetzky A , Tiefenthaler G
Published: June 2004

mAb R73 detects a T cell-specific surface molecule consisting of two disulfide-linked subunits of 40 and 46 kD, respectively, on 97% of peripheral rat T cells, as defined by the OX-52 marker. Of the few OX-52+ R73- cells, none are CD4+ but many express the CD8 antigen known to be present on rat NK cells. mAb R73 is mitogenic for unseparated spleen cells and for purified T cells. In the absence of non-T "accessory cells", stimulation by R73 requires artificial crosslinking of the mAb and is largely dependent on exogenous IL-2. Overnight incubation of purified T cells with crosslinked R73 mAb induces blastoid transformation, IL-2-R expression, and modulation of the R73 antigen. In the rat thymus, mature medullary cells express the R73 determinant at the same level as peripheral T cells, whereas 94% of CD4-CD8- thymocytes are R73-. The major CD4+8+ thymocyte population contains 25% R73- and 70% R73low cells. Thymocytes of the CD4-CD8+OX-44- subpopulation that are the direct precursors of CD4+CD8+ cells display a continuum of R73 antigen density from undetectable to very low levels. We conclude that R73 is most likely directed at a constant determinant of the rat alpha/beta heterodimeric TCR and suggest that CD8+ immature thymocytes are the first cells in the T cell differentiation pathway to express this molecule at their surface.
Antibody recognition of the tumor-specific bcr-abl joining region in chronic myeloid leukemia.
Authors: van Denderen J , Hermans A , Meeuwsen T , Troelstra C , Zegers N , Boersma W , Grosveld G , van Ewijk W
Published: June 2004

Chronic myeloid leukemia (CML) is characterized by the presence of a 210-kD protein (P210bcr-abl) in the cytoplasm of leukemic cells, generated by the reciprocal translocation between chromosome 9 and chromosome 22. Due to this translocation, the abl oncogene is coupled to the bcr gene, forming a new determinant in this protein encoded by the bcr-abl joining region. In the joining region itself, either the bcr exon 2 is coupled to the abl exon 2 (b2-a2), or the bcr exon 3 is coupled to the abl exon 2 (b3-a2). Thus, these joining regions form by definition new tumor-specific determinants in the respective chimeric P210-bcr-abl molecules. This paper addresses the question as to whether these tumor-specific joining regions are exposed on the P210bcr-abl molecule in such a way that antibodies can be generated to detect these sites. To test this possibility a polyclonal antiserum, termed BP-1, was raised against a synthetic peptide representative for the b2-a2 joining region. The reactivity of BP-1 was analyzed in an ELISA system on various synthetic peptides. Peptide inhibition studies showed the presence of antibodies to different parts of the b2-a2 peptide in the polyvalent antiserum. The reactivity of BP-1 was then tested with native P210bcr-abl molecules in various CML cell lines (K562, LAMA-84, and BV173) using a protein kinase assay. In this context, the bcr-abl junctions were first analyzed at the DNA and RNA level. The present study indicates that BP-1 specifically recognizes the b2-a2 junction in native P210bcr-abl. Furthermore, BP-1 clearly discriminates between b2-a2 P210bcr-abl and b3-a2 P210bcr-abl. We conclude that the tumor-specific b2-a2 joining region is antigenically exposed on the native P210bcr-abl molecule.
FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens.
Authors: Klarnet J P , Kern D E , Okuno K , Holt C , Lilly F , Greenberg P D
Published: June 2004

Immunization of C57BL/6 (B6) mice with FBL, a Friend murine leukemia virus (F-MuLV), induces both tumor-specific cytolytic CD8+ (CTL) and lymphokine-producing CD4+ Th that are effective in adoptive therapy of B6 mice bearing disseminated FBL leukemia. The current study evaluated the F-MuLV antigenic determinants expressed on FBL that are recognized by FBL-reactive CD8+ and CD4+ T cells. To identify the specificity of the FBL-reactive CD8+ CTL, Fisher rat embryo fibroblast (FRE) cells transfected with plasmids encoding F-MuLV gag or envelope (env) gene products plus the class I-restricting element Db were utilized. FBL-reactive CTL recognized FRE target cells transfected with the F-MuLV gag-encoded gene products, but failed to recognize targets expressing F-MuLV env. Attempts to generate env-specific CD8+ CTL by immunization with a recombinant vaccinia virus containing an inserted F-MuLV env gene were unsuccessful, despite the generation of a cytolytic response to vaccinia epitopes, implying that B6 mice fail to generate CD8+ CTL to env determinants. By contrast, CD4+ Th clones recognized FRE target cells transfected with env and not gag genes, and immunization with the recombinant vaccinia virus induced an env-specific CD4+ T cell response. These data show that in a Friend retrovirus-induced tumor model in which tumor rejection can be mediated by either CTL or Th, antigens derived from discrete retroviral proteins are predominantly responsible for activation of each T cell subset.
Chromosomal organization of the human major histocompatibility complex class I gene family.
Authors: Koller B H , Geraghty D E , DeMars R , Duvick L , Rich S S , Orr H T
Published: June 2004

17 HLA class I genes have been isolated from the genome of B-lymphoblastoid cell line 721. Sequence analysis and transfection studies indicate that three genes, in addition to those encoding the HLA-A, -B, and -C antigens can direct the synthesis of a class I alpha protein (4, 5, 21). Using gene-specific DNA probes to analyze the presence of restriction fragment-length polymorphisms within a large pedigree and in panel of HLA deletion mutant cell lines, we show here that two of these genes, designated HLA-G and HLA-F, are located on the short arm of chromosome 6 telomeric to the HLA-A locus. The third expressed non-A, -B, and -C class I gene, HLA-E, is located between HLA-A and HLA-C (4). In addition, the remaining 11 class I pseudogenes and gene fragments are localized relative to established markers on chromosome 6p.
Antibody to the murine type 3 complement receptor inhibits T lymphocyte-dependent recruitment of myelomonocytic cells in vivo.
Authors: Rosen H , Milon G , Gordon S
Published: June 2004

We have used the delayed-type hypersensitivity (DTH) response to SRBC or tuberculin to examine the role of the murine type 3 complement receptor in T lymphocyte-dependent inflammatory recruitment. Intravenous injection of 5C6, a CR3-specific rat mAb known to impair myelomonocytic adhesion, divided the DTH to SRBC in actively immunized mice into two phases. The early phase, which lasted 24 h, was characterized by maximal oedema and maximal inflammatory recruitment and was 5C6 inhibitable. The later phase was 5C6 resistant and reached a peak 48 h after antigenic challenge and was superimposable on the declining peak seen in control mice. Passive transfer of reactive T cells mixed with antigen was used to examine the myelomonocytic effector arm of the DTH alone. Both passive transfer of cutaneous DTH to SRBC and passive transfer of the largely monocytic T cell-dependent recruitment to tuberculin in the peritoneal cavity were completely abolished by systemic 5C6 treatment. Injection of 5C6-treated donor leukocytes at the site of passive transfer had no effect. Treatment of donor mice with 5C6 at the time of active immunization did not alter their ability to provide reactive T cells for passive transfer. The myelomonocyte-restricted rat mAb 7/4 and the rapidly cleared F(ab')2 fragment of 5C6 showed no inhibition of the DTH. In all cases, inhibition of footpad swelling correlated with histological evidence of inhibition of myelomonocytic cell recruitment. Peritoneal cell counts after local DTH to tuberculin showed complete inhibition of monocyte recruitment. We conclude that CR3 plays a quantitatively important role in T cell-dependent inflammatory recruitment. This is absolute in passive transfer experiments, but only partial after active immunization. Leukocyte CR3 plays a common role in both immunologically specific and nonspecific inflammatory recruitment and provides a target that could possibly be manipulated to therapeutic advantage.
Production of hematopoietic colony-stimulating factors by human natural killer cells.
Authors: Cuturi M C , Anegón I , Sherman F , Loudon R , Clark S C , Perussia B , Trinchieri G
Published: June 2004

We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.
Mouse thymic virus (MTLV). A mammalian herpesvirus cytolytic for CD4+ (L3T4+) T lymphocytes.
Authors: Morse S S , Valinsky J E
Published: June 2004

Mouse thymic virus (MTLV; ICTV designation murid herpesvirus 3) infects developing T lymphocytes of neonatal mice, causing thymic necrosis and acute immunosuppression. Infected animals shed virus indefinitely. In the present report, two-color flow cytometric analysis of T lymphocyte subpopulations defined by the markers CD4 (L3T4) and CD8 (Lyt-2) was used to determine whether MTLV was lytic for a specific thymocyte population. At peak necrosis (8-11 d after infection), numbers of CD4+8+ cells in the thymus were reduced by 80% or more as compared with controls, and CD4+8- cells were reduced by greater than 98%. The major survivors were CD4-8+ and CD4-8- lymphocytes. These data indicate that the CD4 bearing lymphocyte is a primary target for cytolysis during MTLV infection. Possible parallels between MTLV and a newly described lymphotropic human herpesvirus, human herpesvirus 6 (HHV-6/HBLV), are also suggested.
The tumor-rejection antigens of the 1591 ultraviolet fibrosarcoma. Potential origin and evolutionary implications.
Authors: Linsk R , Watts S , Fischer A , Goodenow R S
Published: June 2004

Previously, we cloned and sequenced the three novel MHC class I genes expressed by the C3H UV fibrosarcoma, 1591. We have extended the analysis of the polymorphic nature of these genes relative to the C3H strain. Scattered nucleotide differences among the tumor genes as compared with the C3H H-2 and Qa sequences make it highly unlikely that the novel tumor genes were generated by recombination between endogenous C3H sequences. Given that two of the tumor clones, A149 and A166, are remarkably similar in amino acid and DNA sequence to H-2Lq and H-2Dq, respectively, we also examined the 1591 RP2 and GUS loci for evidence of polymorphism. Compared with C3H and B10.AKM, 1591 appears to be heterozygous at each of these loci, consistent with an H-2q origin for the two novel 1591 class I genes. Interestingly, the third tumor gene, designated A216, shares certain characteristics with the H-2Ks antigen, reminiscent of the naturally occurring combination of H-2Ks, H-2Dq, and H-2Lq antigens found in some Swiss mouse strains. As a result, we propose that the non-C3H/HeN characteristics displayed by the 1591 tumor point to a non-C3H origin for the novel tumor class I genes of 1591.
Presentation of exogenous protein antigens by dendritic cells to T cell clones. Intact protein is presented best by immature, epidermal Langerhans cells.
Authors: Romani N , Koide S , Crowley M , Witmer-Pack M , Livingstone A M , Fathman C G , Inaba K , Steinman R M
Published: June 2004

The capacity of dendritic cells to present protein antigens has been studied with two MHC class II-restricted, myoglobin-specific, T cell clones. Spleen dendritic cells and cultured epidermal Langerhans cells (LC) presented native myoglobin weakly and often not at all. These same populations were powerful stimulators of allogeneic T cells in the primary MLR. Freshly isolated LC were in contrast very active in presenting proteins to T cell clones but were weak stimulators of the MLR. Both fresh and cultured LC could present specific peptide fragments of myoglobin to the clones. These results suggest that dendritic cells in nonlymphoid tissues like skin can act as sentinels for presenting antigens in situ, their accessory function developing in two phases. First antigens are captured and appropriately presented. Further handling of antigen then is downregulated while the cells acquire strong sensitizing activity for the growth and function of resting T lymphocytes. The potent MLR stimulating activity of cultured epidermal LC and lymphoid dendritic cells probably reflects prior handling of antigens leading to the formation of allogeneic MHC-peptide complexes.
Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis.
Authors: Hameed A , Olsen K J , Lee M K , Lichtenheld M G , Podack E R
Published: June 2004

This study investigates the effect of the purified membrane pore formers, staphylococcal alpha-toxin and CTL perforin, on target cell lysis as measured by 51Cr release and on nuclear damage as measured by DNA degradation and 125IUdR release. Both pore formers cause dose-dependent cell lysis, which is accompanied by DNA release. The ratio of DNA/Cr release depends on the nature of target cell and shows the same pattern as the ratio of release of the two markers reported for CTL-mediated lysis of the same targets. DNA degradation is dependent on the presence of intracellular Ca in the target cell and is totally blocked if Ca is chelated by Quin 2 intracellularly and EGTA extracellularly. DNA degradation, in addition, is inhibited by the lysosomotropic agents NH4Cl, chloroquine, and monensin. rTNF doubles the degree of DNA degradation mediated by alpha-toxin in 3-h assays. We conclude that pore formers alone can mediate DNA degradation. In addition, they may promote the uptake of other factors and thereby accelerate their time course of action. DNA degradation by pore formers requires active target participation in a pathway that is dependent on intracellular Ca and lysosomes. These aspects of target lysis resemble CTL- and NK cell-mediated cytolysis.
Role of N-linked glycans in the interaction between the envelope glycoprotein of human immunodeficiency virus and its CD4 cellular receptor. Structural enzymatic analysis.
Authors: Fenouillet E , Clerget-Raslain B , Gluckman J C , Guétard D , Montagnier L , Bahraoui E
Published: June 2004

gp120 and CD4 are two glycoproteins that are considered to interact together to allow the binding of HIV to CD4+ cells. We have utilized enzymatic digestion by endoglycosidases in order to analyze N-linked carbohydrate chains of these proteins and their possible role in the interaction of gp120 or gp160 with CD4. SDS denaturation was not necessary to obtain optimal deglycosylation of either molecule, but deglycosylation of CD4, nonetheless, depended on the presence of 1% Triton X-100. Endo H and Endo F that cleave high mannose type and biantennary glycans diminish the molecular mass of the glycoproteins from 120 or 160 Kd to 90 or 130 Kd, respectively; but these enzymes had no action on CD4 glycans. Endo F N-glycanase mixture, which acts on all glycan species, including triantennary chains, led to complete deglycosylation of gp120/160 and of CD4. Therefore, probably half of the glycan moieties of gp120/160 are composed of high mannose and biantennary chains, the other half being triantennary species. The carbohydrate structures of CD4 seems to be triantennary chains. To analyze the binding of gp120/160 to CD4, we used a molecular assay in which an mAb (110-4) coupled to Sepharose CL4B allowed the attachment of soluble gp120/160 to the beads; 125I-sCD4 was then added to measure the binding of CD4 to different amounts of gp120/160. Binding to gp160 was not modified when using completely deglycosylated 125I-sCD4, while deglycosylation of gp120 or of gp160 resulted in the decrease of the binding to native CD4 by two- and fivefold, respectively. Native and deglycosylated gp120/160 bound to CD4+ cells with comparable affinities. In addition, deglycosylated gp120 displaced 125I-gp160 binding to CD4+ cells and inhibited fusion of fresh Molt-T4 cells with CEM HIV1- or HIV2-infected cells to the same extent. Taken together, these results indicate that carbohydrates of CD4 and of gp120/160 do not play a significant role in the in vitro interaction between these two molecules.
Class II major histocompatibility complex-restricted T cells specific for a virion structural protein that do not recognize exogenous influenza virus. Evidence that presentation of labile T cell determinants is favored by endogenous antigen synthesis.
Authors: Eisenlohr L C , Hackett C J
Published: June 2004

The contribution of viral infectivity to the expression of MHC class II-restricted T cell determinants was studied. A murine I-Ed-restricted T cell hybridoma recognizing the neuraminidase (NA) glycoprotein of influenza PR8 virus was stimulated strongly by infectious virus but failed to recognize antigen introduced on noninfectious virions. Recognition correlated with the de novo synthesis of viral NA within infected APC. The effectiveness of infectious virus did not depend strictly upon the amount of NA present in cultures, since high NA concentrations could be achieved by addition of nonreplicative virus without being stimulatory for NA-specific T cells. Recognition of a determinant generated only when synthesized in murine host cells was ruled out, since, in high concentration, NA isolated from purified egg-grown virions, even if reduced and alkylated, was recognized by the T hybridoma clone. Isolated NA was recognized when added to pre-fixed APC, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. Data suggest that endogenously synthesized antigen may contribute most significantly to presentation of labile T cell determinants. In addition to NA, recognition of an I-Ed-restricted determinant of the influenza hemagglutinin (HA) molecule, shown previously to have a relatively short half-life on APC surfaces, was enhanced greatly by infectious virus. In contrast, T cell recognition of a more stably expressed I-Ed-restricted site of the same HA polypeptide was only marginally improved on infected APC.
Structural and binding analysis of a two domain extracellular CD2 molecule.
Authors: Sayre P H , Hussey R E , Chang H C , Ciardelli T L , Reinherz E L
Published: June 2004

The 50-kD CD2 (T11) surface glycoprotein on human T lymphocytes and thymocytes plays a critical role in T lineage cell activation and adhesion via its ligand LFA-3. To begin to define structure-function relationships in the extracellular segment of the transmembrane CD2 molecule, we have used a eukaryotic expression system and a CD2 cDNA to produce milligram amounts of recombinant soluble CD2 molecule that corresponds to the two extracellular segment exons. We show that this protein, termed T11ex2, behaves as a monomer in aqueous solution and includes a proteolytically resistant NH2-terminal fragment (domain I) encoded by the first extracellular segment exon. Circular dichroism analysis of T11ex2 demonstrates that its stabilized secondary structure is dependent on the intrachain disulfide bonds present in domain II. The T11ex2 monomer binds directly to the CD2 ligand LFA-3 with a dissociation constant of 0.4 microM. This relatively low affinity implies that cooperative binding resulting from an array of transmembrane CD2 molecules is important to facilitate physiologic T cell adhesion.
An L-arginine-dependent mechanism mediates kupffer cell inhibition of hepatocyte protein synthesis in vitro
Authors: Billiar TR , Curran RD , Stuehr DJ , West MA , Bentz BG , Simmons RL
Published: June 2004

The hepatic failure associated with severe sepsis is characterized by specific, progressive, and often irreversible defects in hepatocellular metabolism (1). Although the etiologic microbe can often be identified, the direct causes and mechanisms of the hepatocellular dysfunction are poorly understood. We have hypothesized that Kupffer cells (KC), which interact with ambient septic stimuli, respond by providing signals to adjacent hepatocytes (HC) in sepsis . Furthermore, we have provided evidence (2, 3) that KC activated by LPS from Gram-negative bacteria can induce profound changes in the function of neighboring HC in coculture. In our model, coculture of either KC (2) or peritoneal macrophages (Mφ)(3) with HC normally promotes HC protein synthesis ([(3)H]leucine incorporation). The addition of LPS or killed Escherichia colt' to such cocultures induces a profound decrease in HC protein synthesis, as well as qualitative changes ([(35)S]methionine, SDS-gel electrophoresis) in protein synthesis without inducing HC death (2, 3) . In this report we show that the inhibition in protein synthesis is mediated via an L-arginine-dependent mechanism.

The metabolism of L-arginine by activated Mφ to substances with cytostatic and even lethal effects on target cells is a relatively recent discovery. After the description by Stuehr and Marletta (4, 5) that LPS- triggered Mφ produced nitrite/nitrate (NO(2)(-)/NO(3)(-)), Hibbs et al. (6, 7) and Iyengar et al. (8) demonstrated that L-arginine was the substrate for the formation of both these nitrogen end products and citrulline. A role for the arginine-dependent mechanism in Mφ tumor cytotoxicity (6, 7) and microbiostatic activity (9) has been suggested. However, the in vivo functions of this novel Mφ mechanism have not yet been defined, but it is possible that there are both physiologic as well as pathologic roles. Our in vitro results raise the possibility that some metabolic responses to microbial invasion maybe partially mediated by the L-arginine-dependent mechanism. What other metabolic responses are affected and the possible pathologic consequences remain to be studied.
Genetic organization of complement receptor-related genes in the mouse.
Authors: Kingsmore S F , Vik D P , Kurtz C B , Leroy P , Tack B F , Weis J H , Seldin M F
Published: June 2004

Using an interspecific cross, gene linkage relationships among members of the murine complement receptor-related genes, C4bp, Cfh, Mcry, and Mcr2, were analyzed by segregation of RFLP in 200 mice. The human homologues of these genes are tightly linked, composing the RCA locus, which maps to human chromosome (Chr.)1q32, within a large linkage group conserved between human Chr.1q21-32 and mouse Chr.1. RFLP associated with C4bp and Cfh map within this conserved linkage group; Cfh is located 9 cM telomeric to C4bp, which is consistent with linkage data for their human homologues. Mcry and Mcr2, while tightly linked, are located outside the conserved group, 40 cM telomeric to C4bp. These data suggest that a translocation or inversion occurred within the RCA family during the evolution of the mouse, defining a breakpoint of this large conserved linkage group.
Molecular cloning of a murine fibronectin receptor and its expression during inflammation. Expression of VLA-5 is increased in activated peritoneal macrophages in a manner discordant from major histocompatibility complex class II.
Authors: Holers V M , Ruff T G , Parks D L , McDonald J A , Ballard L L , Brown E J
Published: June 2004

Human fibronectin receptor (VLA-5) alpha and beta chain probes were used to identify their mouse homologues in a thioglycollate-elicited peritoneal exudate cell cDNA library. Sequence analysis of both alpha and beta chain-related murine clones revealed approximately 90% homology to their human counterparts by both nucleotide and derived amino acid sequence comparisons. Detectable alpha chain transcripts were seen predominantly in total RNA of peritoneal macrophages. beta chain expression, however, was detected at higher levels in lung, heart, brain, and kidney, suggesting the presence of a large murine VLA family similar to the human family. Analysis of levels of expression comparing resting peritoneal macrophages with macrophages elicited using inflammatory stimuli indicated that alpha chain message and surface VLA-5 expression were significantly increased using thioglycollate or Listeria monocytogenes as stimuli to elicit cells. Interestingly, beta chain message was unaffected by these inflammatory stimuli, suggesting that VLA-5 expression is regulated by VLA-5 alpha chain message levels. These results indicate that macrophage VLA-5 expression can be modulated in vivo and may provide an important mechanism by which macrophages are recruited to or adhere to fibronectin in inflammatory foci.
Stroke in Icelandic patients with hereditary amyloid angiopathy is related to a mutation in the cystatin C gene, an inhibitor of cysteine proteases.
Authors: Levy E , Lopez-Otin C , Ghiso J , Geltner D , Frangione B
Published: June 2004

Cystatin C is an inhibitor of lysosomal cysteine proteases and consists of 120 amino acids. A variant of cystatin C lacking the first NH2-terminal residues and having one amino acid substitution at position 68 forms amyloid deposits mainly in the walls of brain arteries, causing fatal strokes in Icelandic patients with familial cerebral hemorrhage secondary to a form of an autosomal dominant amyloidosis. To understand the molecular basis of the genetic defect, the gene encoding cystatin C was isolated from genomic DNA libraries made from normal tissue and the brain of an Icelandic patient with hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). The data indicate that the cystatin C gene encodes a polypeptide of 146 amino acids, of which the first 26 correspond to a secretory peptide signal sequence. The gene contains two intervening sequences that interrupt the coding region at amino acids 55 and 93. Comparison with genes encoding salivary cystatins and kininogen proteins show sequence homology and conservation of exon-intron structure. Except for a mutation in the second exon (CAG instead of CTG in the normal gene, resulting in the substitution of glutamine for a leucine residue), the gene cloned from the brain of the Icelandic patient is identical to the normal cystatin C gene. Thus, HCHWA-I is the first familial type of amyloidosis related to a point mutation in a gene encoding for an inhibitor. The mutation in the structural gene encoding cystatin C appears to be the primary defect in this inherited disorder causing amyloid fibril formation and accumulation followed by cerebral hemorrhage.
Direct observation of the gene organization of the complement C4 and 21-hydroxylase loci by pulsed field gel electrophoresis.
Authors: Dunham I , Sargent C A , Dawkins R L , Campbell R D
Published: June 2004

Pulsed field gel electrophoresis and enzymes that cut genomic DNA infrequently have been used to define large RFLPs at the human C4 loci. With the enzymes BssH II or Sac II, and C4 or 21-hydroxylase DNA probes, it has been possible to observe directly the number of C4 genes present on a haplotype, and also whether the C4 genes are long (6-7-kb intron present) or short (6-7-kb intron absent). Haplotypes that have either two long C4 genes or one long and one short C4 gene generate BssH II fragments of approximately 115 or approximately 105 kb, respectively. Haplotypes that have either a single long or a single short C4 gene generate BssH II fragments of approximately 80 or approximately 70 kb, respectively. This technique has been used to analyze the DNA isolated from PBMC and allows the complete definition of the C4 gene organization of an individual without the need for family studies.
Purification and partial primary sequence of a chemotactic protein for polymorphonuclear leukocytes derived from human lung giant cell carcinoma LU65C cells.
Authors: Suzuki K , Miyasaka H , Ota H , Yamakawa Y , Tagawa M , Kuramoto A , Mizuno S
Published: June 2004

A chemotactic protein for polymorphonuclear leukocytes (lung carcinoma-derived chemotaxin [LUCT]) was purified from culture fluid of the human lung giant cell carcinoma LU65C cells to electrophoretically homogeneous form through five sequential purification steps: DEAE-Sepharose, CM-Sepharose, HPLC on carboxyl-methylated-polyvinylalcohol resin, hydrophobic, and reversed-phase. The molecular mass was determined as approximately 10 kD by SDS-PAGE and isoelectric point was 10.7. The chemotactic activity (ED50 0.75 x 10(-9) M) was sevenfold more potent than that of FMLP (5 X 10(-9) M) and comparable with that of C5a (10(-9) M). NH2-terminal amino acid sequence and amino acid composition of LUCT strongly suggest that it may be closely related to the putative protein encoded by the cDNA clone (3-10C) and almost identical with a part of sequence of the chemotactic factor derived from stimulated human leukocytes in the 6th to 32nd, but not the NH2-terminal 5 amino acids. These results indicate that the carcinoma cells produce LUCT without any added stimulant and suggest that the previously isolated chemotactic monokines may correspond to des(1-5) of LUCT in the NH2-terminal region.
Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins.
Authors: Brett J , Gerlach H , Nawroth P , Steinberg S , Godman G , Stern D
Published: June 2004

Endothelium is an important target of tumor necrosis factor/cachectin (TNF), a central mediator of the host response in endotoxemia and Gram-negative sepsis. In this report, TNF is shown to increase the permeability of endothelial cell monolayers to macromolecules and lower molecular weight solutes by a mechanism involving a pertussis toxin-sensitive regulatory G protein. Within 1-3 h of exposure to TNF (5 nM), changes in cell shape/cytoskeleton occurred that led to disruption of monolayer continuity with the formation of intercellular gaps. Correlated with these structural changes was an increase in endothelial permeability to macromolecular and lower molecular weight tracers; time-dependent, reversible increases in passage of these tracers, evident by 1-3 h, were observed after addition of TNF to cultures. Perturbation of barrier function by TNF also depended on the dose of TNF added being half-maximal by approximately 0.4 nM. Only a brief exposure (15 min) of TNF to endothelium was required to induce an increase in permeability, and this was not prevented by the presence of cycloheximide or actinomycin D. Preincubation of monolayers with pertussis toxin blocked in parallel TNF-induced increased passage of solutes and cell shape/cytoskeletal perturbation, indicating the close correlation between these changes in endothelial cell function. In contrast, pertussis toxin did not alter TNF-induced modulation of two endothelial cell coagulant properties. These data provide evidence for two intracellular pathways of TNF action that are distinguishable by pertussis toxin and provide insight into a mechanism underlying loss of solute from the intravascular space mediated by TNF: alteration in endothelial cell barrier function.
Induction of pigmentation in mouse fibroblasts by expression of human tyrosinase cDNA.
Authors: Bouchard B , Fuller B B , Vijayasaradhi S , Houghton A N
Published: June 2004

A distinguishing characteristic of cells of the melanocyte lineage is the expression of the melanosomal enzyme tyrosinase that catalyzes the synthesis of the pigment melanin. A tyrosinase cDNA clone, designated BBTY-1, was isolated from a library constructed from the pigmented TA99+/CF21+ melanoma cell line SK-MEL-19. Expression of BBTY-1 in mouse L929 fibroblasts led to synthesis and expression of active tyrosinase, and, unexpectedly, to stable production of melanin. Melanin was synthesized and stored within membrane-bound vesicles in the cytoplasm of transfected fibroblasts. BBTY-1 detected a 2.4-kb mRNA transcript in nine of nine pigmented, tyrosinase-positive melanoma cell lines. Tyrosinase transcripts of the same size and abundance were detected in a subset (three of eight) of nonpigmented, tyrosinase-negative melanoma cell lines, suggesting that post-transcriptional events are important in regulating tyrosinase activity. Two melanocyte antigens, recognized by mAbs TA99 and CF21, that are specifically located within melanosomes and are coexpressed with tyrosinase activity, did not react with transfected mouse fibroblasts expressing human tyrosinase, supporting the conclusion that these antigenic determinants are distinct from the tyrosinase molecule coded for by BBTY-1.
Role of CD4 in thymocyte selection and maturation.
Authors: Zuñiga-Pflücker J C , McCarthy S A , Weston M , Longo D L , Singer A , Kruisbeek A M
Published: June 2004

We examined the possible role of CD4 molecules during in vivo and in vitro fetal thymic development. Our results show that fetal thymi treated with intact anti-CD4 mAbs fail to generate CD4 single-positive T cells, while the generation of the other phenotypes remains unchanged. Most importantly, the use of F(ab')2 and Fab anti-CD4 mAb gave identical results, i.e., failure to generate CD4+/CD8- T cells, with no effect on the generation of CD4+/CD8+ T cells. Since F(ab')2 and Fab anti-CD4 fail to deplete CD4+/CD8- in adult mice, these results strongly argue that the absence of CD4+/CD8- T cells is not due to depletion, but rather, is caused by a lack of positive selection, attributable to an obstructed CD4-MHC class II interaction. Furthermore, we also observed an increase in TCR/CD3 expression after anti-CD4 (divalent or monovalent) mAb treatment. The TCR/CD3 upregulation occurs in the double-positive population, and may result from CD4 signaling after mAb engagement, or may be a consequence of the blocked CD4-class II interactions. One proposed model argues that the CD3 upregulation occurs in an effort to compensate for the reduction in avidity or signaling that is normally provided by the interaction of the CD4 accessory molecule and its ligand. As a whole, our findings advocate that CD4 molecules play a decisive role in the differentiation of thymocytes.
Clonal diversity in the B cell repertoire of patients with X-linked agammaglobulinemia.
Authors: Anker R , Conley M E , Pollok B A
Published: June 2004

Ig protein and mRNA expression was examined in a collection of 18 monoclonal EBV-transformed B cell lines derived from five patients with X-linked agammaglobulinemia (XLA). A diversity of H and L chain isotypes were synthesized by these lines: the majority (12 lines) expressed mu kappa chains, while mu lambda (two lines), gamma kappa (one), gamma lambda (one), delta lambda (one), and alpha kappa (one) isotype expression was also observed. For all the mu kappa-producing XLA B cell lines, the mu and kappa mRNA transcripts were of native size, and sequence analysis across the regions of VHDJH and V kappa J kappa gene joining showed that Ig gene rearrangements occurred in a typical manner. A variety of VHDJH and V kappa J kappa gene rearrangements were observed, not only within the set of mu kappa+ XLA B cells as a whole, but also among the cell lines derived from single patients. Southern blot analysis for genomic Ig H chain gene rearrangements was done to fully assess the extent of clonal heterogeneity among multiple mu kappa+ XLA B cell lines derived from two patients; all the B cell lines possessed distinct gene rearrangement patterns demonstrating their clonal unrelatedness. Our findings indicate that the B cell repertoire in individual XLA patients is clonally diverse and that it is unlikely that the defect in B cell differentiation in XLA is the result of inefficient or ineffective rearrangement of Ig H or L chain genes. Rather, this study provides support for the idea that the XLA defect relates to a more generalized cellular function, such as regulating the proliferation and/or clonal expansion of cells of the B lymphoid lineage.
Antibody recognition of the type 14 pneumococcal capsule. Evidence for a conformational epitope in a neutral polysaccharide.
Authors: Wessels M R , Kasper D L
Published: June 2004

Oligosaccharides consisting of one or more tetrasaccharide repeating units were derived from the capsular polysaccharide of type 14 pneumococcus (Pn14) by endo-beta-galactosidase digestion. The relative affinity of anticapsular antibody binding to derivative oligosaccharides of different chain lengths was measured in a Pn 14 ELISA inhibition assay. The concentration of inhibiting antigen required to achieve 50% inhibition of IgG binding increased progressively from 5.6 x 10(-4) M to 7.0 x 10(-11) M as the inhibiting saccharide chain length increased from 1 tetrasaccharide repeating unit to 2,500 repeating units. These data indicate that antibodies directed against the Pn14 polysaccharide recognize a conformational epitope fully expressed only in high molecular weight forms of the antigen. Similar results were found for inhibition of Fab fragment binding, suggesting that recognition of the conformational epitope is largely dependent on the intrinsic affinity of the Fab combining region. Unlike previously reported polysaccharides for which conformational epitopes have been described, the Pn14 polysaccharide does not contain negatively charged residues, indicating that expression of conformational determinants is not limited to acidic polysaccharides. Antibody recognition of conformational epitopes may be a common mechanism by which the host immune response discriminates between bacterial polysaccharides and host oligosaccharides of similar structure.
Inflammatory properties of recombinant tumor necrosis factor in rabbit skin in vivo.
Authors: Rampart M , De Smet W , Fiers W , Herman A G
Published: June 2004

We have investigated the ability of recombinant TNF (mouse and human) to produce acute inflammatory lesions in an established experimental model of inflammation. Upon intradermal injection in rabbit skin, TNF, in amounts as low as 3 x 10(-14) mol/site, was found to be very potent at inducing local neutrophil accumulation and neutrophil-dependent oedema formation, thereby fulfilling two important criteria to be considered as an inflammatory mediator. Our findings further indicate that the pro-inflammatory properties of TNF are probably more related to its immediate stimulatory effects on neutrophils rather than to its slow (protein biosynthesis-dependent effects on endothelial cells. Our data thus show that very low amounts of mouse and human recombinant TNF can initiate an acute inflammatory reaction in vivo in rabbit skin and that TNF is able to evoke two of the four cardinal signs of inflammation.
Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule.
Authors: Lanier L L , Testi R , Bindl J , Phillips J H
Published: June 2004

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.
Phenotypic and functional alteration of CD4+ T cells after antigen stimulation. Resolution of two populations of memory T cells that both secrete interleukin 4.
Authors: Hayakawa K , Hardy R R
Published: June 2004

Phenotypic and functional alteration of murine CD4+ T cells after antigenic stimulation was studied using two anti-T cell mAbs recently described that define four distinct T cell subsets. Activation of T cells resulted in the permanent loss of 3G11 expression. However, two phenotypically distinct memory T cell populations were established depending on the system used; whereas those for anti-KLH antibody response were enriched in the fraction expression 6C10 (Fr. III), memory T cells for the allogeneic MLR lacked such expression (Fr. IV). Furthermore, successive stimulation with antigen in vitro resulted in secretion of IL-4 without detectable IL-2. This alteration of phenotype and interleukin secretion was also demonstrable when starting with 3G11+6C10- cells (Fr. I), the fraction that secretes IL-2 exclusively upon activation.
Circulating interleukin 6 during a continuous infusion of tumor necrosis factor and interferon gamma.
Authors: Brouckaert P , Spriggs D R , Demetri G , Kufe D W , Fiers W
Published: June 2004

Plasma samples obtained from patients receiving a 24-h continuous infusion of human recombinant (hr)TNF or a combination of two overlapping, 24-h continuous infusions of hrIFN-gamma and hrTNF were analyzed for IL-6 in a sensitive bioassay. A transient appearance of circulating IL-6 was observed with peak levels between 3 and 6 h after the start of the hrTNF infusion. These peak levels correlated quite well with the dose of hrTNF administered (r = 0.86; p less than 0.001). The maximal value observed was 27.5 ng/ml IL-6 in a sample of a patient receiving 545 micrograms/m2 hrTNF. The combination of hrIFN-gamma (200 micrograms/m2) and hrTNF in the infusions resulted in higher IL-6 levels than a comparable dose of hrTNF alone. A maximal value of 23.5 ng/ml IL-6 was observed in a patient receiving 205 micrograms/m2 hrTNF. No IL-6 was found in the plasma of patients during the 12-h infusion with hrIFN-gamma alone, except for two borderline samples.
Independent regulation of Ca2+ entry and release from internal stores in activated B cells.
Authors: Gelfand E W , MacDougall S L , Cheung R K , Grinstein S
Published: June 2004

Addition of crosslinking antibody to B lymphocytes results in a rapid rise in cytoplasmic-free Ca2+ ([Ca2+]i) due to release of Ca2+ from internal stores and uptake of Ca2+ across the plasma membrane. Inositol 1,4,5-trisphosphate is believed to mediate the release of internal Ca2+ stores and has also been proposed to mediate extracellular Ca2+ entry. We have compared the properties of these two pathways for Ca2+ mobilization by dissociating the [Ca2+]i changes in ligand-activated human B cells after loading of the cells with the Ca2+ chelator BAPTA. In the present paper we show that: (a) the sustained increase in [Ca2+]i is due to increased unidirectional influx of external [Ca2+]i; (b) entry of extracellular Ca2+, but not release of internal stores, is sensitive to the transmembrane potential; and (c) entry of extracellular Ca2+, but not release of internal stores, is inhibited by increasing [Ca2+]i. These findings suggest that the permeation pathways mediating the translocation of Ca2+ across the plasma membrane and endoplasmic reticulum membrane are not identical.
Acquired resistance to Listeria monocytogenes is mediated by Lyt-2+ T cells independently of the influx of monocytes into granulomatous lesions.
Authors: Mielke M E , Niedobitek G , Stein H , Hahn H
Published: June 2004

Facultative intracellular bacteria induce specific T cell responses of both the CD4+ and the CD8+ subsets. The immunohistological study of the tissue responses to Listeria monocytogenes in T cell subset-depleted, Listeria-primed mice revealed that CD4+ cells not only represent the predominant lymphocyte population in granulomatous lesions but mediate the attraction and accumulation of blood-borne monocytes into inflammatory foci. On the other hand, CD8+ T cells are able to mediate protection in the absence of CD4+ T cells, invading monocytes, and granulomatous inflammation, and therefore appear to activate resident macrophages for listericidal activity.