bluuebunny/crossref_metadata_2025 huggingface dataset:
Naive and memory T cells show distinct pathways of lymphocyte recirculation.
Authors: Mackay C R , Marston W L , Dudler L
Published: June 2004

In this report, we have addressed two questions concerning immunological memory: the way in which naive and memory T cells recirculate through the body, and the intrinsic rate of division within the naive and memory populations. We identified naive and memory T cells in sheep by their cell surface phenotype and their ability to respond to recall antigen. Memory T cells were CD2hi, CD58hi, CD44hi, CD11ahi, and CD45R-, as pertains in man. T cells that crossed from blood to the tissues of the hind leg and accumulated in the popliteal afferent lymph were all of memory phenotype. Conversely, T cells in efferent lymph, 90% of which entered the lymph node (LN) via high endothelial venules (HEV), were mostly of the naive phenotype (CD2lo, CD58lo, CD44lo, CD11alo, and CD45R+). The marked enrichment of these two phenotypes in different recirculatory compartments indicated that memory T cells selectively traffic from blood to peripheral tissues to LN (via afferent lymph), whereas naive T cells selectively traffic from blood to LN (via HEV). We argue that the differential use of these two recirculation pathways probably optimizes lymphocyte interactions with antigen. The nonrandom distribution of T cell subsets in various recirculatory compartments may be related to the relative proportion of memory cells in each subset. In particular, gamma/delta T cells in blood were almost exclusively of memory phenotype, and accumulated preferentially in afferent, but not in efferent, lymph. Finally, using the bromo-deoxyuridine labeling technique, we found that at least a sizeable proportion of memory T cells, whether in blood or afferent lymph, were a dividing population of cells, whereas naive T cells were a nondividing population. This result supports an alternative model of lymphocyte memory that assumes that maintenance of memory requires persistent antigenic stimulation.
Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.
Authors: Idzerda R L , March C J , Mosley B , Lyman S D , Vanden Bos T , Gimpel S D , Din W S , Grabstein K H , Widmer M B , Park L S
Published: June 2004

IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this report we describe the isolation and functional expression of a human IL-4-R cDNA. When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein. After transfection into a murine T cell line, the cDNA encodes a protein that binds human IL-4 with high affinity and can confer responsiveness to human IL-4. The predicted extracellular domain of the IL-4-R exhibits significant amino acid sequence homology with the beta subunit of the IL-2-R (p75), and the receptors for IL-6, erythropoietin, and prolactin. These receptors comprise a novel superfamily with extracellular domains characterized by four conserved cysteine residues and a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane region.
Expression of the Fc-receptor for IgE (Fc epsilon RII, CD23) on alveolar macrophages in extrinsic allergic alveolitis.
Authors: Pforte A , Breyer G , Prinz J C , Gais P , Burger G , Häussinger K , Rieber E P , Held E , Ziegler-Heitbrock H W
Published: June 2004

Expression of the Fc receptor for IgE (Fc epsilon R) was analyzed on alveolar macrophages (AM) in 10 patients with extrinsic allergic alveolitis (EAA) compared with 10 patients with sarcoidosis and to 6 apparently healthy controls. By using the anti-Fc epsilon RII mAb M-L25 in immunocytochemistry experiments, we found that greater than 60% of AM in 10 of 10 patients with EAA were strongly positive, as evidenced by visual analysis in light microscopy and by cytometry. By contrast, no significant staining was detected in sarcoidosis or in controls with either method. Similar results were obtained when Fc epsilon R were identified with preformed immune complexes consisting of NIP-specific human/mouse chimeric IgE antibody plus NIP-ovalbumin. Furthermore, greater than 60% of AM in patients with EAA stained positive for IgE, demonstrating that endogenous IgE is bound to the AM. Our data suggest that IgE antibodies bound to Fc epsilon RII on AM may be involved in pathophysiology of extrinsic allergic alveolitis by activation of the AM after binding of allergen to the cell surface IgE. Furthermore, with the clearcut pattern of Fc epsilon RII expression in extrinsic allergic alveolitis it may be possible to use CD23 antibodies for differential diagnosis of inflammatory lung disease.
The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product.
Authors: Vijayasaradhi S , Bouchard B , Houghton A N
Published: June 2004

The gp75 antigen is an abundant intracellular glycoprotein expressed in melanosomes of human pigmented melanocytes and melanomas. IgG antibodies in sera of a patient with metastatic melanoma have been shown to immunoprecipitate gp75, suggesting that immunological tolerance against gp75 can be broken. The mouse mAb TA99, which specifically recognizes gp75, was used to isolate and purify the antigen. Amino acid sequences of three internal peptides were determined from the purified gp75 polypeptide. cDNA clones were isolated by screening with oligonucleotides based on these peptide sequences. The gp75 peptides and cDNA had approximately 90% identity with, respectively, the derived amino acid and nucleotide sequences of a mouse gene that maps to the b (brown) locus. The brown locus determines coat color in the mouse, suggesting that gp75 regulates or influences the type of melanin synthesized.
Human epidermal T cells predominantly belong to the lineage expressing alpha/beta T cell receptor.
Authors: Foster C A , Yokozeki H , Rappersberger K , Koning F , Volc-Platzer B , Rieger A , Coligan J E , Wolff K , Stingl G
Published: June 2004

The epidermis of clinically normal-appearing human skin harbors a phenotypically heterogeneous population of T lymphocytes (TCs), the majority of which are CD2+/CD3+/CD5+ "memory" cells, but in an unactivated state, and express the TCR-alpha/beta. In contrast to murine skin, only a very minor subpopulation of CD3+ cells in the human epidermis bears the TCR-gamma/delta. Epidermal TCs primarily are distributed along the rete ridges in the basal keratinocyte layer and are often in close apposition to Langerhans cells (LCs). These TCs were propagated from epidermal cell suspensions after stimulation with TC activating agents (Con A, rIL-1, rIL-2), then evaluated for phenotypic features and TCR diversity. Similar to the in situ situation, most were CD4-/CD8+/TCR-alpha/beta+. In addition, two cultures contained TCR-gamma/delta+ cells; one of these determined to be an adherent CD4-/CD8+ population. Epidermal TCs were significantly (p less than 0.0001) more abundant in the sole than in the other body regions examined (i.e., 40 vs. 7 CD3+ cells/linear centimeter of epidermis) and seemed to have a particular affinity for the acrosyringial epithelium of eccrine sweat ducts. Moreover, the sole usually contained a greater number of CD8+ relative to CD4+ TCs, whereas the epidermal CD4/CD8 ratio in the trunk and extremities was quite variable, although the trend also was towards a slightly larger percentage of CD8+ cells. Collectively, our data suggest that the volar epidermis has a unique microenvironment which is responsible for both the higher density of TCs, preferentially CD8+, and lower number of LCs. This study has not only provided evidence for significant regional variability in the human epidermal TC population of normal skin, but also strengthens the concept for skin-associated lymphoid tissues (SALT), whereby memory TCs recirculate back to the epidermis and interact with resident antigen-presenting cells (i.e., LC).
LAG-3, a novel lymphocyte activation gene closely related to CD4.
Authors: Triebel F , Jitsukawa S , Baixeras E , Roman-Roman S , Genevee C , Viegas-Pequignot E , Hercend T
Published: June 2004

We have identified a novel human gene of the Ig superfamily, designated LAG-3. Expression of this gene is undetectable in resting PBL, while it is found (a 2-kb message) in activated T and NK cells. The LAG-3 gene includes eight exons; the corresponding cDNA encodes a 498-amino acid membrane protein with four extracellular IgSF domains. The first one belongs to the V-SET; it is particular since it includes an extra loop in the middle of the domain and an unusual intrachain disulphide bridge. The three other domains belong to the C2-SET. Strong internal homologies are found in the LAG-3 molecule between domains 1 and 3, as well as between domains 2 and 4. It is therefore likely that LAG-3 has evolved by duplication of a pre-existing gene encoding a two IgSF-domain structure. The compared analysis of LAG-3 and CD4, with respect to both their peptidic sequence as well as their exon/intron organization, indicated that the two molecules are closely related. This point is strengthened by the finding that both genes are located on the distal part of the short arm of chromosome 12.
Preferential nuclear compartmentalization of endogenous mink cell focus-forming-related retroviral transcripts.
Authors: Gourley M F , Krieg A M , Steinberg A D
Published: June 2004

Endogenous mink cell focus-forming (MCF)-like retroviral sequences in the murine genome are stable, inherited sequences analogous to other chromosomal genes. As such, it is thought that they are transcribed and translated in a manner analogous to other genes. However, when the SL12.4 CD4-, CD8- thymoma cell line was studied for nuclear/cytoplasmic distribution of endogenous MCF-related transcripts, there was a nuclear predominance. The great majority of full-length 8.4-kb endogenous MCF-related transcripts were nuclear. Even the smaller, spliced 3.0-kb transcripts were at least as prominent in the nucleus as the cytoplasm, whereas cellular RNA was 80% cytoplasmic and other cellular transcripts were represented in the cytoplasm to a much greater extent than the nucleus. Size cannot fully account for the nuclear presence of MCF-related endogenous transcripts, because the 3.0-kb MCF transcripts occurred in the nucleus to a much greater relative extent than 3.8-kb c-myb transcripts. These studies point to retroviral-like structures of these transcripts as influencing their intracellular compartmentalization.
Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae.
Authors: Berish S A , Mietzner T A , Mayer L W , Genco C A , Holloway B P , Morse S A
Published: June 2004

This report describes the cloning and sequencing of the major iron-regulated protein (termed Fbp) of Neisseria gonorrhoeae strain F62. Attempts to identify recombinants expressing the Fbp using specific antibody proved unsuccessful. Therefore, an alternative cloning strategy using oligonucleotide probes derived from NH2-terminal and tryptic fragments of this protein was used to identify short fragments of the gene. Using this methodology, the gene encoding the precursor of Fbp was cloned on three separate overlapping fragments and sequenced, and the amino acid sequence was deduced. These data were unambiguously confirmed by the known NH2-terminal amino acid sequence and were supported by the sequences from tryptic fragments that lie outside of this region. Using oligonucleotide probes, we were unable to obtain clones encoding the potential regulatory region of this protein. Therefore, the technique of inverse polymerase chain reaction was used to amplify a fragment containing an additional 200 bp. This fragment was cloned and sequenced and found to contain a consensus ribosome binding site and potential -10 and -35 sequences. Hybridization analysis of genomic DNA from gonococcal strain F62 indicated that only a single copy of the Fbp gene exists per genome. These results complement the biochemical characterization of the Fbp expressed by gonococci and further suggest that it has a role in iron-acquisition.
Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice.
Authors: Van Zant G , Holland B P , Eldridge P W , Chen J J
Published: June 2004

We have studied contributions to hematopoiesis of genetically distinct stem cell populations in allophenic mice. Chimeras were made by aggregating embryos of inbred strains known to differ with respect to stem cell population kinetics. One partner strain (DBA/2) has previously been shown to normally have a stem cell (CFU-S) population of which 24% are in S-phase of the cell cycle, whereas the homologous population of the other partner strain (C57BL/6) was characterized by having only 2.6% in cycle (7). Contributions of the chimeric stem cell population to mature blood cell pools were studied throughout the life of the mice and intrinsic differences in stem cell function and aging were reflected in dynamic patterns of blood cell composition. The DBA/2 stem cell population was eclipsed by stem cells of the C57BL/6 genotype and, after 1.5-3 yr, the hemato-lymphoid composition of 22 of 27 mice studied for this long had shifted by at least 25 percentage points toward the C57BL/6 genotype. 8 of the 27 had hematolymphoid populations solely of C57BL/6 origin. To test whether or not a population of stem cells with an inherently higher cycling rate (DBA/2) might have a competitive advantage during repopulation, we engrafted allophenic marrow into lethally irradiated (C57BL/6 x DBA/2)F1 recipients. DBA/2 hematopoiesis was predominant early, far outstripping its representation in the marrow graft. Perhaps as a consequence of inherently greater DBA/2 stem cell proliferation, the populations of developmentally more restricted precursor populations (CFU-E, BFU-E, CFU-GM, CFU-GEMM) showed an overwhelming DBA/2 bias in the first 2-3 mo after engraftment. However, as in the allophenic mice themselves during the aging process, the C57BL/6 genotypic representation was ascendant over the subsequent months. The shift toward C57BL/6 genotype was first documented in the marrow and spleen precursor cell populations and was subsequently reflected in the circulating, mature blood cells. Bone marrow-derived stromal cell cultures from engrafted mice were studied and genotypic analyses showed donor representation in stromal cell populations that reflected donor hematopoietic contributions in the same recipient. Results from these studies involving two in vivo settings (allophenic mice and engraftment by allophenic marrow) are consistent with the notion that a cell autonomous difference in stem cell proliferation confers on one population a competitive repopulating advantage, but at the expense of longevity.
Retinol is essential for growth of activated human B cells.
Authors: Buck J , Ritter G , Dannecker L , Katta V , Cohen S L , Chait B T , Hämmerling U
Published: June 2004

When EBV-transformed human B cells are removed from conventional cell cultures, washed, and seeded at a low cell density in serum-free medium, their growth potential is greatly diminished. Fresh serum restores the growth of low density B cell cultures. We have traced this restorative effect to an essential factor present in the lipid fraction of serum and have identified it as all-trans retinol. The identification is based on the close similarities of the factor isolated from serum with authentic all-trans retinol as revealed by mass spectrometry, HPLC chromatography, and the ability to stimulate the growth of lymphoblastoid cells in the bioassay. Retinol is active at concentrations equal to its concentration in serum. Retinol is also a requirement for growth in suspension cultures at cell densities of 3 x 10(5)/ml. Cells removed at any time from such exponentially growing cultures and transferred to retinol-free medium cease to grow and consequently die, whereas in the continued presence of retinol, cell growth is unabated. All-trans retinal can substitute for retinol, but retinoic acid fails to stimulate the growth of lymphoblastoid cells at physiological concentrations. Normal human B lymphocytes also require retinol as a costimulator of proliferation after activation by anti-mu antibody or Staphylococcus aureus (Cowan strain) bacteria. In serum, retinol is bound to retinol-binding protein, which in turn forms a complex with prealbumin. Accordingly, we find that B cells respond to retinol bound to its physiological serum carrier, retinol-binding protein. In conclusion, human B cells are critically dependent for optimal growth in cell culture on an external supply of retinol.
Intracerebroventricular injection of interleukin 1 induces high circulating levels of interleukin 6.
Authors: De Simoni M G , Sironi M , De Luigi A , Manfridi A , Mantovani A , Ghezzi P
Published: June 2004

IL-1 is known to have a central role in the induction of acute-phase response, and some of its activities (including induction of some acute-phase proteins) were reported to be mediated by an induction of IL-6. Administration to rats of 200 ng of human rIL-1 by intracerebroventricular injection resulted in a more marked induction of circulating IL-6 than the same dose of IL-1 administered systemically (intravenously or intraperitoneally). Induction of serum IL-6 by centrally administered IL-1 was also observed in hypophysectomized or adrenalectomized rats, suggesting that activation of the hypothalamus-pituitary-adrenal axis is not essential for this effect of IL-1. IL-6 induction was also observed after pretreatment with indomethacin, indicating that the effect was dissociated from the pyrogenic activity of IL-1. Induction of IL-6 by a central action could represent a novel pathway in IL-1-induced acute-phase response.
Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice.
Authors: Heremans H , Van Damme J , Dillen C , Dijkmans R , Billiau A
Published: June 2004

The involvement of cytokines in the pathogenesis of a generalized, Shwartzman-like lethal inflammatory response to bacterial lipopolysaccharides (LPS) was studied by testing the ability of cytokines or neutralizing anticytokine antibodies to modify the course of the syndrome. The reaction was elicitable in non-SPF NMRI mice by two consecutive injections of S. marcescens LPS: a first injection in the footpad, followed after 24 h by an intravenous dose; the size and route of the preparatory LPS dose were found to be critical. Treatment with mAbs against IFN-gamma was found to completely prevent the reaction. Treatment with IFN-gamma on the other hand, rendered the mice more sensitive to elicitation of the reaction. In contrast, systemic administration of IFN-alpha/beta exerted a desensitizing effect. The role of endogenous cytokines in the pathogenesis of this generalized Shwartzman reaction was also documented by a study of the cytokine levels in the serum of the mice. In comparisons between mice given lethal and nonlethal induction schedules, a good correlation was found between mortality rates and height of IFN or TNF levels, but no correlation was seen with IL-6 levels. Also, in mice that were protected by anti-IFN-gamma antibody, serum IFN and TNF were undetectable, whereas IL-6 levels were as high as in unprotected mice. These data provide evidence that among the cytokines that govern the inflammatory response to LPS, endogenous IFN-gamma occupies a key position. These findings therefore also open perspectives for clinical application of IFN-gamma antagonists.
Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meningitidis. Synthetic peptides define the epitopes responsible for serosubtype specificity.
Authors: McGuinness B , Barlow A K , Clarke I N , Farley J E , Anilionis A , Poolman J T , Heckels J E
Published: June 2004

The previously determined nucleotide sequence of the porA gene, encoding the class 1 outer membrane protein of meningococcal strain MC50, has been used to clone and sequence the porA gene from two further strains with differing serosubtype specificities. Comparison of the predicted amino acid sequences of the three class 1 proteins revealed considerable structural homology with major variation confined to two discrete regions (VR1 and VR2). The high degree of structural homology between the sequences gave predicted secondary structures that were almost identical, with the variable domains located in hydrophilic regions that are likely to be surface located and hence accessible to antibody binding. The predicted amino acid sequences have been used to define the epitopes recognized by mAbs with serosubtype specificity. A series of overlapping decapeptides spanning each of the class 1 protein sequences have been synthesized on solid-phase supports and probed with mAbs. Antibodies with P1.16 and P1.15 subtype specificity reacted with sequences in the VR2 domain, while antibodies with P1.7 subtype specificity reacted with sequences in the VR1 domain. Further peptides have been constructed to define the minimum epitopes recognized by each antibody. Thus we have been able to define linear peptides on each class 1 protein molecule that are responsible for subtype specificity and that represent targets for a protective immune response.
An immunoglobulin light chain from a lupus-prone mouse induces autoantibodies in normal mice.
Authors: Puccetti A , Koizumi T , Migliorini P , André-Schwartz J , Barrett K J , Schwartz R S
Published: June 2004

Autoantibodies against the 70-kD U1 RNP nucleoprotein autoantigen and DNA were elicited in normal BALB/c mice with a purified Ig light chain. This light chain, derived from a lupus-prone MRL-lpr/lpr mouse, has two distinctive properties: it contains an idiotypic marker recognized by a monoclonal MRL-lpr/lpr anti-snRNP autoantibody, and the amino acid sequence of its third hypervariable region (CDR3) is homologous to a sequence in an antigenic region of the 70-kD U1 RNP polypeptide. The results demonstrate that an Ig idiotype that mimics an autoantigen can induce autoimmunization.
Viruses as therapeutic agents. II. Viral reassortants map prevention of insulin-dependent diabetes mellitus to the small RNA of lymphocytic choriomeningitis virus.
Authors: Oldstone M B , Ahmed R , Salvato M
Published: June 2004

Nonobese diabetic (NOD) mice are the experimental prototype of type 1 insulin-dependent diabetes mellitus (IDDM). These mice develop a characteristic autoimmune lesion in the pancreatic islets of Langerhans, where infiltrating lymphocytes destroy beta cells, resulting in hypoinsulinemia, hyperglycemia, ketoacidosis, and death. This IDDM, which closely resembles that in humans, is prevented by infecting NOD mice with particular strains of lymphocytic choriomeningitis virus (LCMV), including Armstrong 53b, Traub, WE, and Pasteur. In contrast, the LCMV Armstrong 53b variant, Clone 13, fails to abort IDDM. Hence, although Clone 13 establishes a persistent infection that endures throughout the life spans of NOD mice, their hyperglycemia, hypoinsulinemia, and lymphocytic infiltration into the islets of Langerhans still occur. Genetic reassortant viruses generated between the IDDM therapeutic strain of LCMV Pasteur and the nontherapeutic variant, LCMV Clone 13, were used to treat NOD mice. By using such reassortants and both parental strains of virus to infect NOD mice, the prevention of IDDM was mapped to the S RNA segment of LCMV Pasteur.
Analysis of somatic mutations in kappa transgenes.
Authors: Hackett J , Rogerson B J , O'Brien R L , Storb U
Published: June 2004

We have examined the nature and localization of somatic mutations in three kappa transgenes cloned from IgG-secreting hybridomas. All of the mutations identified were single base substitutions. Mutations were localized to the variable (V) region and its flanking sequences. In every case, the nuclear matrix association region, kappa enhancer, and C gene were spared. These data indicate that the rearranged kappa gene contains the necessary sequences for targeting of the mutation process, and suggest that the observed localization of mutations to the V region reflects the inherent specificity of this mutation process.
Phagocytosis of tumor cells by human monocytes cultured in recombinant macrophage colony-stimulating factor.
Authors: Munn D H , Cheung N K
Published: June 2004

Macrophages and cultured human monocytes can mediate efficient antibody-dependent cytotoxicity (ADCC) against human tumor cells using monoclonal antibodies (mAbs). The mechanism of this killing is usually assumed to involve secreted factors (reactive oxygen intermediates, tumor necrosis factor, or other cytotoxic factors) leading to target cell lysis. In this study, we present evidence that phagocytosis of intact target cells is the principal mechanism of antitumor cytotoxicity in our in vitro model of ADCC by cultured monocytes. Human monocytes cultured in recombinant human macrophage colony-stimulating factor ingested up to 100% of fluorochrome-labeled melanoma and neuroblastoma target cells, in the presence of an appropriate antitumor mAb. Electron microscopy demonstrated phagocytosis of intact tumor cells by cultured monocytes during ADCC. All of the radionuclide in radiolabeled target cells was taken up by monocytes during phagocytosis. By preventing the release of radioisotope tracers, phagocytosis thus prevents the detection of this very efficient form of cytotoxicity by most conventional assays.
The role of polymorphic HLA-DR beta chain residues in presentation of viral antigens to T cells.
Authors: Karr R W , Yu W , Watts R , Evans K S , Celis E
Published: June 2004

The relative importance of 11 polymorphic positions in the HLA-DR7 beta 1 chain in T cell recognition of foreign antigens was investigated using transfectants expressing mutant DR7 beta 1 chains as APC for five rabies virus-specific T cell clones. The results indicate that multiple amino acids, located in both the beta-strands and alpha-helix of DR7 beta 1 in the model of a class II molecule, are involved in DR7-restricted T cell recognition of these antigens. Many of the substitutions appeared to reduce the affinity of an antigenic peptide for the mutant DR7 molecules but did not prevent binding. The heterogeneity of responses of the three G-specific T cell clones to presentation of the G11.3 peptide by several of the mutant DR7 molecules indicates that the T cell receptor (TCR) of each these clones requires a different view of the G11.3/DR7 complex and raises the possibility that the G11.3 peptide may bind to the DR7 molecule in more than one conformation.
Parallel evolution of antibody variable regions by somatic processes: consecutive shared somatic alterations in VH genes expressed by independently generated hybridomas apparently acquired by point mutation and selection rather than by gene conversion.
Authors: Wysocki L J , Gefter M L , Margolies M N
Published: June 2004

We identified, in independently generated hybridoma antibodies, blocks of shared somatic alterations comprising four consecutive amino acid replacements in the CDR2s of their heavy chain variable regions. We found that the nucleotide sequences encoding the shared replacements differed slightly. In addition, we performed genomic cloning and sequencing analyses that indicate that no genomic sequence could encode the block of shared replacements in any one of the antibodies and thus directly serve as a donor by a recombinational process. Finally, in a survey of other somatically mutated versions of the same heavy chain variable gene, we found several examples containing one, two, or three of the shared CDR2 mutations in various combinations. We conclude that the shared somatic alterations were acquired by several independent events. This result, and the fact that the antibodies containing the four shared mutations were elicited in response to the same antigen and are encoded by the same VH and VK gene segments, suggests that an intense selection pressure has fixed the shared replacements by favoring the clonal expansion of B cells producing antibodies that contain them. The basis of this selection pressure is addressed elsewhere (Parhami-Seren, B., L. J. Wysocki, M. N. Margolies, and J. Sharon, manuscript submitted for publication).
Differentiation of normal human pre-B cells in vitro.
Authors: Villablanca J G , Anderson J M , Moseley M , Law C L , Elstrom R L , LeBien T W
Published: June 2004

The differentiation of surface Ig- pre-B cells into surface Ig+ B cells is a critical transition in mammalian B cell ontogeny. Elucidation of the growth factor requirements and differentiative potential of human pre-B cells has been hampered by the absence of a reproducible culture system that supports differentiation. Fluorescence-activated cell sorting and magnetic bead depletion were used to purify fetal bone marrow CD10+/surface mu- cells, which contain 60-70% cytoplasmic mu+ pre-B cells. CD10+/surface mu- cells cultured for 2 d were observed to differentiate into surface mu+ cells. Analysis by Southern blotting provided direct evidence that rearrangement of kappa light chain genes occurs in culture, and flow cytometric analysis revealed the appearance of surface Ig+ B cells expressing mu/kappa or mu/lambda. Unexpectedly, the kappa/lambda ratio in differentiated cells was the inverse of what is normally observed in adult peripheral blood. Differentiation occurs in the absence of exogenous growth factors or cytokines, suggesting that a stimulus-independent differentiative inertia might characterize pre-B cells in vivo. Future use of this model will facilitate our understanding of normal and abnormal human pre-B cell differentiation.
Superantigenicity of streptococcal M protein.
Authors: Tomai M , Kotb M , Majumdar G , Beachey E H
Published: June 2004

M proteins that define the serotypes of group A streptococci are powerful blastogens for human T lymphocytes. The mechanism by which they activate T cells was investigated and compared with the conventional T cell mitogen phytohemagglutinin, and the known superantigen staphylococcal enterotoxin B. Although major histocompatibility complex (MHC) class II molecules are required for presentation, there is no MHC restriction, since allogeneic class II molecules presented the bacterial protein to human T cells. Type 5 M protein appears to bind class II molecules on the antigen-presenting cells and stimulate T cells bearing V beta 8 sequences. Our results indicate that this streptococcal M protein is a superantigen and suggest a possible mechanism of its role in the pathogenesis of the postinfectious autoimmune sequelae.
Age-related factors in cyclosporine-induced syngeneic graft-versus-host disease: regulatory role of marrow-derived T lymphocytes.
Authors: Fischer A C , Hess A D
Published: June 2004

The present studies have evaluated the effect of age on the induction of syngeneic graft-versus-host disease (SGVHD) after syngeneic bone marrow transplantation (BMT) and cyclosporine (CsA) therapy. The results clearly document an inverse correlation of age with the incidence of SGVHD. Virtually a 100% incidence of SGVHD occurs in Lewis rats when syngeneic BMT and CsA therapy are started when the animals are 4 wk of age. Thereafter, there is a dramatic decline in the incidence of SGVHD with the increasing age of the animals. Although the age of the recipient was important, the most significant effect was the age of the marrow donor. Marrow from animals 6 mo of age was virtually incapable of eliciting SGVHD after BMT and CsA therapy. Furthermore, mixing the marrow from mature and immature animals resulted in a decreased incidence of SGVHD, implicating a regulatory effect present in the marrow from older rats. This regulatory effect was due to the presence of mature T cells in the marrow from animals 6 mo of age. Despite the fact that marrow from young animals possesses mature T lymphocytes, this regulatory activity was absent, suggesting that the host resistance mediated by T lymphocytes develops as the animal ages. These data further implicate the importance of a host resistance mechanism in preventing the induction of SGVHD with CsA, which appears to be mediated by the clonal inactivation of autoreactive cells.
Hybrid T cell receptor genes formed by interlocus recombination in normal and ataxia-telangiectasis lymphocytes.
Authors: Lipkowitz S , Stern M H , Kirsch I R
Published: June 2004

In this paper, using polymerase chain reaction (PCR), we demonstrated the occurrence of hybrid genes formed by interlocus recombination between T cell receptor gamma (TCR-gamma) variable (V) regions and TCR-beta joining (J) regions in the peripheral blood lymphocytes (PBL) from normal individuals and patients with ataxia-telangiectasia (AT). Sequence analysis of the PCR-derived hybrid genes confirmed that site-specific V gamma-J beta recombination had occurred and showed that 10 of 23 genomic hybrid genes maintained a correct open reading frame. By dilution analysis, the frequency of these hybrid genes was 8 +/- 1/10(5) cells in normal PBL and 587 +/- 195/10(5) cells in AT PBL. These frequencies and the approximately 70-fold difference between the normal and AT samples are consistent with previous cytogenetic data examining the occurrence of an inversion of chromosome 7 in normal and AT PBL. We also demonstrated expression of these hybrid genes by PCR analysis of first-strand cDNA prepared from both normal and AT PBL. Sequence analysis of the PCR-amplified transcripts showed that, in contrast to the genomic hybrid genes, 19 of 22 expressed genes maintained a correct open reading frame at the V-J junction and correctly spliced the hybrid V-J exon to a TCR-beta constant region, thus allowing translation into a potentially functional hybrid TCR protein. Another type of hybrid TCR transcript was found in a which a rearranged TCR-gamma V-J exon was correctly spliced to a TCR-beta constant region. This form of hybrid gene may be formed by trans-splicing. These hybrid TCR genes may serve to increase the repertoire of the immune response. In addition, studies of their mechanism of formation and its misregulation in AT may provide insight into the nature of the chromosomal instability syndrome associated with AT. The mechanism underlying hybrid gene formation may be analogous to the mechanism underlying rearrangements between putative growth-affecting genes and the antigen receptor loci, which are associated with AT lymphocyte clones and lymphoid malignancies.
The major 85-kD surface antigen of the mammalian form of Trypanosoma cruzi is encoded by a large heterogeneous family of simultaneously expressed genes.
Authors: Kahn S , Van Voorhis W C , Eisen H
Published: June 2004

Trypanosoma cruzi is an obligate intracellular protozoan parasite. The parasite mammalian stage surface antigens exhibit extensive antigenic diversity. We have characterized a family of T. cruzi genes that code for a polymorphic set of 85-kD surface antigens, the SA85-1 antigens. The family contains greater than 100 genes and pseudogenes, of which a minimum of nine are transcribed. The gene family is expressed in the mammalian stage only. A subset of the gene family is present in two telomere-linked copies in the genome. Telomere linkage of other expressed SA85-1 genes has not been demonstrated. We have shown that at least three members of the SA85-1 gene family encode antigens at the surface of the mammalian stage of the parasite. Interestingly, these three antigens are expressed on all the trypanosomes examined. This suggests that T. cruzi simultaneously expresses a large repertoire of similar, but diverse antigens at its surface. Thus, T. cruzi exhibits extensive antigenic diversity in a system unique from that of African trypanosomes, perhaps reflecting its intracellular niche.
Oxygen-dependent modulation of erythropoietin mRNA levels in isolated rat kidneys studied by RNase protection.
Authors: Ratcliffe P J , Jones R W , Phillips R E , Nicholls L G , Bell J I
Published: June 2004

Using oligonucleotide primers complementary to conserved regions in the mouse erythropoietin (Epo) gene, a portion of the rat Epo gene was amplified by the polymerase chain reaction to produce a probe suitable for assay of rat Epo mRNA by RNAse protection. The assay, which has sufficient sensitivity to measure to Epo mRNA in unstimulated rat kidneys, was used to demonstrate high amplitude in vitro modulation of Epo mRNA levels in response to changes in perfusate flow rate and oxygen tension in isolated kidneys, thus providing clear evidence that all the necessary events linking changes in oxygen delivery to the modulation of Epo mRNA levels can occur intrarenally.
In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody.
Authors: Heyman B , Wiersma E J , Kinoshita T
Published: June 2004

BALB/c mice were injected intravenously with three different monoclonal antibodies (mAbs) specific for complement receptor 1 (CR1). Two of the mAbs crossreacted with CR2. 24 h later, the mice were immunized with horse erythrocytes or keyhole limpet hemocyanin (KLH), and the primary antibody response was measured. One of the anti-CR antibodies, 7G6, suppressed greater than 99% of the direct plaque-forming cell response against horse red blood cells (HRBC). The same antibody markedly suppressed the serum antibody responses to both HRBC and KLH. To be optimally suppressive, the mAb had to be injected before suboptimal concentrations of antigen. The other two complement receptor-specific antibodies had very moderate, if any, effects on the antibody response. 7G6 was able to downregulate CR1 and CR2 on the surface of B cells and, in addition, to inhibit rosette formation with C3d-coated sheep erythrocytes (EC3d). One of the antibodies with a weak effect downregulated only CR1. The other downregulated both CR1 and CR2, although not as efficiently as 7G6, and was unable to inhibit EC3d rosette formation. We conclude that the reason 7G6 is outstanding in its suppressive capacity is that it is the only mAb tested that functionally blocks CR2. The data suggest that CR2 is of crucial importance in the initiation of a normal antibody response to physiological concentrations of antigen.
Human eosinophils express transforming growth factor alpha.
Authors: Wong D T , Weller P F , Galli S J , Elovic A , Rand T H , Gallagher G T , Chiang T , Chou M Y , Matossian K , McBride J
Published: June 2004

Transforming growth factor alpha (TGF-alpha) is a pleuripotential cytokine with diverse biological effects, including the ability to influence the proliferation of normal cells or neoplastic epithelial cells. Eosinophils are a subset of granulocytes that normally enter the peripheral tissues, particularly those beneath gastrointestinal, respiratory, and urogenital epithelium, where they reside in close proximity to the epithelial elements. In this study, we demonstrate that the great majority of eosinophils infiltrating the interstitial tissues adjacent to two colonic adenocarcinomas and two oral squamous cell carcinomas labeled specifically by in situ hybridization with a 35S-riboprobe for human TGF-alpha (hTGF-alpha). No other identifiable leukocytes in these lesions contained detectable hTGF-alpha mRNA. We also examined leukocytes purified from a patient with the idiopathic hypereosinophilic syndrome. 80% of these eosinophils, but none of the patient's neutrophils or mononuclear cells, were positive for hTGF-alpha mRNA by in situ hybridization, and 55% of these eosinophils were positive by immunohistochemistry with a monoclonal antibody directed against the COOH terminus of the mature hTGF-alpha peptide. Finally, the identification of the purified eosinophil-associated transcript as hTGF-alpha was confirmed by polymerase chain reaction product restriction enzyme analysis followed by Southern blot hybridization. In contrast to eosinophils from the patient with hypereosinophilic syndrome, the peripheral blood eosinophils from only two of seven normal donors had detectable TGF-alpha mRNA and none of these eosinophils contained immunohistochemically detectable TGF-alpha product. Taken together, these findings establish that human eosinophils can express TGF-alpha, but suggest that the expression of TGF-alpha by eosinophils may be under microenvironmental regulation. Demonstration of TGF-alpha production by tissue-infiltrating eosinophils and the eosinophils in the hypereosinophilic syndrome identifies a novel mechanism by which eosinophils might contribute to physiological, immunological, and pathological responses.
Mechanism of formation of human IgE-binding factors (soluble CD23): III. Evidence for a receptor (Fc epsilon RII)-associated proteolytic activity.
Authors: Letellier M , Nakajima T , Pulido-Cejudo G , Hofstetter H , Delespesse G
Published: June 2004

There is mounting evidence that Fc epsilon RII (CD23) and its soluble fragments (IgE-binding factors [BFs] or soluble CD23) have pleiotropic activities. IgE-BFs are formed mainly by the proteolytic cleavage of surface Fc epsilon RII; they are first released as 37- and 33-kD unstable molecules that are subsequently transformed into 25-kD IgE-BFs. In this study, purified and radioiodinated 37-kD IgE-BFs as well as 45-kD Fc epsilon RII were used as substrates to identify the proteases leading to the formation of 25-kD IgE-BFs. These substrates generate 25-kD IgE-BFs when incubated with several Fc epsilon RII-bearing cells, including CHO1-7 cells (transfected with Fc epsilon RII cDNA); by contrast Fc epsilon RII- cells, including CHO control cells, have no effect. Highly purified unlabeled native 37-kD and recombinant 29-kD IgE-BFs also cleave labeled 45-kD Fc epsilon RII into 25-kD IgE-BFs. The proteolytic activity of these purified IgE-BFs is specifically removed by immunoprecipitation with an antibody against IgE-BFs. These data strongly suggest that Fc epsilon RII and some of its soluble fragments play an active role in the proteolytic mechanism generating IgE-BFs. They are supported by the observation that IgE-BFs released by CHO1-7 cells are cleaved exactly at the same sites as B cell-derived IgE-BFs. Taken collectively, the results are compatible with an autoproteolytic process.
Constitutive expression of parathyroid hormone-related protein gene in human T cell leukemia virus type 1 (HTLV-1) carriers and adult T cell leukemia patients that can be trans-activated by HTLV-1 tax gene.
Authors: Watanabe T , Yamaguchi K , Takatsuki K , Osame M , Yoshida M
Published: June 2004

Adult T cell leukemia (ATL) is associated with human T cell leukemia virus type 1 (HTLV-1) infection, and almost all ATL patients have the complication of hypercalcemia. To understand the mechanism of the high incidence of hypercalcemia in ATL, we studied the expression of a parathyroid hormone-related protein (PTHrP) gene that has been proposed as a causative factor of hypercalcemia in some solid tumors. The polymerase chain reaction coupled with reverse transcription of mRNA was applied to RNA from peripheral blood mononuclear cells. Cells from all 13 ATL patients examined showed abundant expression of the PTHrP gene, while cells from uninfected normal subjects did not. Significant expression of PTHrP gene was also detected in HTLV-1 carriers without any symptoms and in patients with HTLV-1-associated myelopathy or tropical spastic paraparesis. PTHrP mRNA levels correlated with the number of infected cells that were estimated by the integrated HTLV-1 DNA. These results suggest that HTLV-1-infected cells are expressing the PTHrP gene. This concept was further supported by the finding that the HTLV-1 trans-activator, the tax gene product, caused trans-activation of the PTHrP gene promoter linked to the CAT gene. These observations might explain the general expression of the PTHrP gene in ATL patients and the high incidence of hypercalcemia in ATL.
T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages.
Authors: Guidos C J , Danska J S , Fathman C G , Weissman I L
Published: June 2004

To identify the maturational stage(s) during which T cell receptor (TCR)-mediated positive and negative selection occurs, we followed the development of CD4+8- and CD4-8+ T cells from TCRlo CD4+8+ thymic blasts in the presence of different positive and negative selecting (major histocompatibility complex or Mls) elements. We describe novel lineage-committed transitional intermediates that are TCRmed CD4+8lo or TCRmed CD4lo8+, and that show evidence of having been positively selected. Furthermore, negative selection is not evident until after cells have attained one of the TCRmed transitional phenotypes. Accordingly, we propose that negative selection in normal mice occurs only after TCRlo CD4+8+ precursors have been positively selected into either the CD4 or CD8 lineage.
A distinct wave of human T cell receptor gamma/delta lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production.
Authors: Krangel M S , Yssel H , Brocklehurst C , Spits H
Published: June 2004

The rearrangement and expression of human T cell receptor (TCR)-gamma and -delta gene segments in clonal and polyclonal populations of early fetal and postnatal human TCR-gamma/delta thymocytes were examined. The data suggest that the TCR-gamma and -delta loci rearrange in an ordered and coordinated fashion. Initial rearrangements at the TCR-delta locus join V delta 2 to D delta 3, and initial rearrangements at the TCR-gamma locus join downstream V gamma gene segments (V gamma 1.8 and V gamma 2) to upstream J gamma gene segments associated with C gamma 1. These rearrangements are characterized by minimal junctional diversity. At later times there is a switch at the TCR-delta locus such that V delta 1 is joined to upstream D delta gene segments, and a switch at the TCR-gamma locus such that upstream V gamma gene segments are joined to downstream J gamma gene segments associated with C gamma 2. These rearrangements are characterized by extensive junctional diversity. Programmed rearrangement explains in part the origin of discrete subpopulations of peripheral blood TCR-gamma/delta lymphocytes that have been defined in previous studies. In addition, cytokine production by early fetal and postnatal TCR-gamma/delta thymocyte clones was examined. Fetal thymocyte clones produced significant levels of IL-4 and IL-5 following stimulation, whereas postnatal thymocyte clones did not produce these cytokines. Thus, these cell populations may represent functionally distinct subsets as well.
Requirement for CD4+ cells in resistance to Pneumocystis carinii pneumonia in mice.
Authors: Harmsen A G , Stankiewicz M
Published: June 2004

The importance of CD4+ cells in resistance to Pneumocystis carinii (PC) in PC-susceptible severe combined immunodeficient (SCID) mice that were made resistant to PC by immunocompetent spleen cell transfer, and in conventional PC-resistant mice, was investigated. SCID mice with naturally acquired PC pneumonia (PCP) were given infusions of spleen cells from immunocompetent donors. This reconstitution caused the recipients to resolve their PCP. Treatment of reconstituted SCID mice with anti-CD4 monoclonal antibodies (mAbs) to deplete them of CD4+ cells eliminated their ability to resolve PCP, whereas treating them with anti-CD8 mAb to deplete CD8+ cells had no effect. The findings indicate, therefore, that resistance to PCP in immunologically reconstituted SCID mice is dependent on CD4+ cells. To determine whether CD4+ cells enable conventional mice to resist PCP, B6D2 mice were treated with anti-CD4 mAb to deplete them of CD4+ cells in an attempt to induce PCP. After 10-11 wk of treatment, these mice developed progressive PCP. Taken together, these results indicate that loss of CD4+ cells predisposes mice to PC infection.