bluuebunny/crossref_metadata_2025 huggingface dataset:
The expression of a gamma interferon-induced protein (IP-10) in delayed immune responses in human skin.
Authors: Kaplan G , Luster A D , Hancock G , Cohn Z A
Published: June 2004

Our knowledge of the induction of new molecules by IFN-gamma has led to the characterization of IP-10 and the preparation of a monospecific, polyclonal antibody. Using this reagent we have now examined inflammatory states occurring in human skin and used immunocytochemical staining for the expression of both Ia and IP-10 determinants. After evoking a delayed-type response to purified protein derivative of tuberculin (PPD), we noted the presence of IP-10 in dermal macrophages and endothelial cells. Intense staining of the basal layer of epidermal keratinocytes was prominent at 41 h, and by 1 wk the entire epidermis was staining. The comparison of the amount of IP-10 secreted by keratinocytes vs. macrophages, fibroblasts, and endothelial cells revealed that keratinocytes were by far the major producers of this molecule. The expression of Ia occurred in conjunction with IP-10. The injection of rIFN-gamma mimicked many of the features of the PPD response, including the expression of both Ia and IP-10 by epidermal keratinocytes. Coexpression was also found in the natural lesions of tuberculoid leprosy and cutaneous Leishmaniasis. However, it was absent in lepromatous leprosy, a state where activated T lymphocytes are not present. We suggest that the local production of IFN-gamma by T cells of the dermal infiltrate induces IP-10 formation in both the dermis and epidermis. IP-10 and Ia then serve as specific markers of immune IFN and its possible influence on effector cells of the cell mediated immune response.
Generation of continuous large granular lymphocyte lines by interleukin 2 from the spleen cells of mice infected with Moloney leukemia virus. Involvement of interleukin 3.
Authors: Hattori M , Sudo T , Iizuka M , Kobayashi S , Nishio S , Kano S , Minato N
Published: June 2004

Continuous cell lines could be reproducibly established by culturing spleen cells from adult mice injected with MLV-producer cells or directly infected with Mo-MLV with rIL-2, whereas the culture of normal splenic cells with rIL-2 induced only transient and limited proliferation resulting in no such lines. All of the lines showed morphological characteristics as LGL with Thy-1+,Lyt-1-,L3T4-,Lyt-2-,AsGM1+,FcR gamma+ phenotype without exception, and most of them exhibited typical NK-patterned cytotoxicity. Analysis of reverse transcriptase activity of the culture supernatants as well as Southern hybridization of the DNA from the lines using an Mo-MLV-specific cDNA probe indicated no evidence of retroviral replication or proviral integration, suggesting that the generation of cell lines reflected a reactive process and viral infection was not directly responsible. It was subsequently revealed that Thy-1+,Lyt-1+,Lyt-2- spleen cells from mice infected with Mo-MLV in vivo spontaneously produced surprising amounts of IL-3 in vitro, leading to the possibility that IL-3 was responsible for the generation of lines. The possibility was directly supported by the observation that continuous lines with identical characteristics could be generated completely in vitro by sequential stimulation with rIL-3 and rIL-2 from normal spleen cells without any involvement of Mo-MLV. The C beta gene of TCR was shown to be rearranged in all the lines examined, indicating the LGL lines were all genetically committed to T cell lineage. Unlike the situation in normal splenic populations expanded by rIL-2, where the expression of IL-2-R was progressively lost, constitutive expression of high-affinity-IL-2-R was observed in all the lines and thus, this was considered to explain the unlimited proliferation of them in response to rIL-2 alone. These results suggested the probable role of IL-3 in the regulation of growth and differentiation of a set of LGL committed to T cell lineage. The possible implications of the phenomenon in the regulation of hematopoiesis as well as in the control of Mo-MLV-induced leukemogenesis were discussed.
DNA receptor dysfunction in systemic lupus erythematosus and kindred disorders. Induction by anti-DNA antibodies, antihistone antibodies, and antireceptor antibodies.
Authors: Bennett R M , Kotzin B L , Merritt M J
Published: June 2004

The ability of sera from patients with SLE and similar connective tissue diseases to induce dysfunction of the receptor for DNA was studied. All SLE and MCTD sera studied resulted in marked inhibition of DNA receptor binding. Furthermore, the sera from a subgroup of patients with other rheumatic diseases and a surprisingly high percentage of asymptomatic relatives of SLE patients exhibited a similar effect. The humoral factors causing this defect were shown to be of at least three reactivities: (a) antibodies to DNA, (b) antibodies to histones, and (c) antibodies to the DNA receptor itself. The reactivity of anti-DNA and antihistone antibodies is dependent upon intact cell-surface DNA, and reconstitution experiments suggest that antihistone antibodies are reactive with histones complexed to this DNA, which in turn is bound to the DNA receptor. Cells with an antibody-induced DNA receptor defect are unable to bind DNA; the subsequent inability to degrade DNA may have important consequences in diseases such as SLE in which DNA-anti-DNA immune complexes are of pathogenetic significance.
Suggested mechanism for the selective excretion of glucosylated albumin. The effects of diabetes mellitus and aging on this process and the origins of diabetic microalbuminuria.
Authors: Kowluru A , Kowluru R , Bitensky M W , Corwin E J , Solomon S S , Johnson J D
Published: June 2004

In previous studies in the Sprague-Dawley rat, Williams and coworkers reported the phenomenon of selective urinary excretion of glucosylated albumin (editing, i.e., the percent glucosylation of urinary albumin is more than that of plasma albumin) by the mammalian kidney. Ghiggeri and coworkers subsequently found that the extent of editing is reduced in human diabetics. Moreover, the reduction in editing in diabetes correlates inversely with levels of microalbuminuria. We also find reduction in the extent of editing in diabetic humans. We find a striking inverse correlation not only with the magnitude of microalbuminuria but also with the extent of plasma albumin glucosylation. In contrast, we found little correlation between the reduction in editing and the duration of diabetes in human subjects. Stz induced diabetes in the Sprague-Dawley rat is associated with a striking and rapid reduction in editing which develops virtually with the same kinetics exhibited by the appearance of hyperglycemia. This loss of editing is rapidly reversed by daily administration of insulin but not by aldose reductase inhibitors. Mannitol infusion in anesthetized Wistar rats resulted in an increase in urine volume, GFR, and microalbuminuria, and was also accompanied by a marked reduction in editing. This reduction was rapidly reversed by a cessation of mannitol infusion. We propose here that glucosylated albumin (in contrast to unmodified albumin) is not reabsorbed by the proximal tubule, and thus, is preferentially excreted in the urine. We postulate that the increase in GFR which emerges as a consequence of increased plasma osmolality in diabetes mellitus delivers more albumin to the proximal tubule than can be reabsorbed. This results in a dilution of excreted glucosylated albumin molecules by excreted unmodified albumin, which appears as the early microscopic albuminuria of diabetes. Paradoxically, the fall in apparent editing is accompanied by an absolute increase in the total quantity of glucosylated albumin excreted. In contrast, we found that editing of glucosylated albumin by the normal kidney is found to gradually decline as a function of age without the appearance of microalbuminuria. This suggests that a different mechanism operates to produce the loss of editing seen with aging in man, and as clearly (but in a shorter absolute time intervals) in the Fischer-344 rat.
Beta transforming growth factors are potential regulators of B lymphopoiesis.
Authors: Lee G , Ellingsworth L R , Gillis S , Wall R , Kincade P W
Published: June 2004

Members of the transforming growth factor beta (TGF-beta) family of polypeptides were found to be potent in vitro inhibitors of kappa light chain expression on normal bone marrow-derived and transformed cloned pre-B cells, and of the maturation of these cells to mitogen responsiveness. The inhibition by TGF-beta was selective in that Ia expression was not blocked. Together with the observations that LPS, IL-1, NZB serum factors, IL-4, and IFN-gamma preferentially induced either kappa or Ia, or both, on a pre-B cell line, these results further suggest that acquisition of Ig and class II molecules is independently controlled by different antagonists as well as agonists. In addition, kappa chain induction by IFN-gamma does not appear to be as sensitive to TGF-beta downregulation as that stimulated by other factors tested, and this raises the possibility that activation of the same gene may result from different transmembrane signaling pathways. In contrast to the inhibitory effects of TGF-beta on kappa acquisition by pre-B cells and on kappa increase after exposure of mature B cells to LPS, as measured by kappa RNA levels and/or surface fluorescence, no inhibition was observed on unstimulated spleen B cells or on two cloned B cell lines that constitutively produce kappa. Thus, TGF-beta may function during specific stages of B cell differentiation by inhibiting initiation of, or increased transcription of Ig genes, and therefore, may be an important negative regulator of B lymphopoiesis. It is the first natural substance found to have this effect.
Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.
Authors: Brüggemann M , Williams G T , Bindon C I , Clark M R , Walker M R , Jefferis R , Waldmann H , Neuberger M S
Published: June 2004

Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications.
Activation and growth of colony-stimulating factor-dependent cell lines is cell cycle stage dependent.
Authors: London L , McKearn J P
Published: June 2004

Hematopoietic cell development is regulated by a series of growth factors that are progressively restricted in their biological activity. IL-3 is a multi-lineage growth factor that supports the growth and differentiation of progenitor cells belonging to multiple lineages. However, the mechanism by which IL-3 induces proliferation and differentiation of these cells is not completely understood. In this report, we have used two IL-3-dependent cell lines, FDC-P1 (a myeloid progenitor) and F15.12 (a lymphoid progenitor) to investigate IL-3-mediated growth and differentiation. When either FDC-P1 or FL5.12 cells are deprived of IL-3, greater than 90% of all cells accumulate in the G0 phase of the cell cycle. Upon readdition of IL-3, the cells will reenter the active phases of the cell cycle. Therefore, IL-3 can act as both a competence (G0----G1) factor, and a progression (G1----M) factor for hematopoietic precursor clones. FDC-P1 cells can also proliferate in response to granulocyte/macrophage colony-stimulating factor (G/M-CSF) and IL-4 (B cell stimulatory factor 1 [BSF-1]). However, resting (G0) FDC-P1 cells have lost their ability to grow in response to both G/M-CSF and IL-4, even though both factors can induce a G0----G1 transition. Therefore, G/M-CSF or IL-4 behave as progression factors among certain IL-3-responsive clones, and in those cases only in defined points in the cell cycle. Both IL-4 and G/M-CSF can maintain long-term growth of FDC-P1 cells. Upon removal of factor for 24 h, these clones accumulate in the G1 phase of the cell cycle and do not appear to enter G0 even after 36 h of factor deprivation. Therefore, cells maintained in G/M-CSF or IL-4 have altered growth requirements compared with the IL-3-dependent lines from which they were derived. The ability of various hematopoietic growth factors to regulate cell cycle progression in IL-3-dependent cell lines is dependent not only upon the lineage from which these cells were derived, but also the phase of the cell cycle in which those cells reside. The consequences of these interactions dictate the manner by which various clones will respond to CSFs and whether the cells will grow and/or differentiate.
Somatically mutated forms of a major anti-p-azophenylarsonate antibody variable region with drastically reduced affinity for p-azophenylarsonate. By-products of an antigen-driven immune response?
Authors: Manser T , Parhami-Seren B , Margolies MN , Gefter ML
Published: June 2004

The pivotal role played by antigen in the clonal selection of B cells for initial participation in an immune response is well established. Antigen selective mechanisms ensure that antigen-binding antibodies are produced during all stages of the immune response. However, antibodies that lack specificity for the immunogen might also be produced during the course of an antigen-driven immune response . It has been suggested that, through idiotype-antiidiotype network interactions within the immune system, production of antibodies that lack specificity for the immunogen but that share idiotopes with antigen-binding antibodies could result (1). In addition, data obtained by a number of investigators suggest that somatic mutation of antibody V region genes occurs at a rate of 10(-3)/basepair/cell division in B cells participating in an immune response (2, 3). One outcome of such V region structural alteration could be antibodies that lack, or have drastically reduced affinity for the immunogen . We sought to identify and characterize some of the antibody by-products of the antigen-driven immune response that are expected to be created by the mechanisms described above.
Inhibition of suppressor T lymphocytes by murine interferon beta.
Authors: Sahasrabudhe D M
Published: June 2004

We have presented data showing that IFN-beta at 1,000 U/mouse i.v. inhibits the generation of Ts-mediated tolerance to dinitrofluorobenzene (DNFB) and abrogates the transfer of suppression by Ts. We have also presented data showing that IFN-beta up to 10,000 U/mouse i.v. has no adverse effect on sensitization and elicitation. IFN-beta appears to be a suitable agent for evaluation as an adjunct in the immunotherapy of Ts inducing tumors.
An intermediate cell in thymocyte differentiation that expresses CD8 but not CD4 antigen.
Authors: Paterson D J , Williams A F
Published: June 2004

Thymocytes of CD4-,CD8+,OX-44- phenotype have been shown to be an intermediate of thymopoiesis that give rise to cells of CD4+, CD8+, OX-44- normal cortical thymocyte phenotype both in vitro and in vivo during thymic regeneration.
Isolation and expression of cDNA clones encoding a human receptor for IgG (Fc gamma RII).
Authors: Stuart S G , Trounstine M L , Vaux D J , Koch T , Martens C L , Mellman I , Moore K W
Published: June 2004

We have cloned and expressed a cDNA encoding a human receptor for IgG (Fc gamma R) from the monocyte cell line U937. The deduced structure is a 35-kD transmembrane protein with homology to the mouse Fc[gamma 2b/gamma 1] receptor amino acid sequence of approximately 60% in the extracellular domain. The signal sequence is homologous to the mouse Fc gamma R alpha cDNA clone, while the transmembrane domain shares homology with mouse Fc gamma R beta cDNAs. The cytoplasmic domain is apparently unique. The extracellular domain shows significant homology to proteins of the Ig gene superfamily, including the human c-fms protooncogene/CSF-1 receptor. Mouse Ltk- cells transfected with the human Fc gamma R cDNA express a cell-surface receptor that selectively binds human IgG and is recognized by the anti-Fc gamma RII mAb IV.3. Antibodies against peptides derived from the human Fc gamma R sequence specifically stain U937 cells, but not an Fc gamma RII-bearing B-lymphoblastoid cell line (Daudi). These results identify the human Fc gamma RII as the homologue of mouse Fc[gamma 2b/gamma 1] R, and provide evidence for heterogeneity of Fc gamma RII expressed on monocytes and B cells.
Human cytotoxic T lymphocytes evoked by group A streptococcal M proteins.
Authors: Dale J B , Beachey E H
Published: June 2004

Purified group A streptococcal M proteins were used to stimulate peripheral blood lymphocytes from normal adult volunteers. The activated lymphocytes were cytotoxic against cultured human heart cells, as well as liver cells, fibroblasts, and K562 cells, but showed only minimal cytotoxicity against several animal cell types. The cytotoxic activity evoked by type 5 M protein was dose and time dependent. Rabbit antisera against pep M5 that contained heart-crossreactive antibodies partially inhibited cytotoxicity against heart cells, but had no effect on other target cells, suggesting that a fraction of the effector lymphocytes may be recognizing M protein-crossreactive cell surface antigens. All of the cytotoxic activity was recovered from a CD3+ population of lymphocytes obtained from a fluorescence-activated cell sorter, and CD4+ and CD8+ cells were also cytotoxic. M protein-responsive T cell clones were generated that showed specificity for heart and K562 cells, in addition to clones that were cytotoxic against both cell lines. Our data show that streptococcal M protein evokes cytotoxic T lymphocytes against multiple human but not animal target cells. Some of the effector cells may be specific for cultured myocardial cells, but their role in the pathogenesis of rheumatic carditis will require further studies of lymphocytes from patients with acute rheumatic fever and carditis.
Aging and arteriosclerosis. The increased proliferation of arterial smooth muscle cells isolated from old rats is associated with increased platelet-derived growth factor-like activity.
Authors: McCaffrey T A , Nicholson A C , Szabo P E , Weksler M E , Weksler B B
Published: June 2004

In vivo studies have suggested that the aorta from an old animal responds to injury with an exaggerated proliferation of smooth muscle cells (SMCs) compared with the response of this aorta from a young animal. In this study we compared proliferation of SMCs derived from uninjured old (less than 19 mo) and young (3-4 mo) rat aortas. Old SMCs grew more rapidly than young SMCs in the presence of medium containing competence factors (10% FCS or platelet-derived growth factor [PDGF]) as well as in their absence (2% PDS or serum-free media) as determined both by a short-term thymidine incorporation assay and by cell counts. Lysates prepared from old SMCs that had been grown in the absence of serum or PDGF stimulated proliferation of target cells more than lysates prepared from young SMCs; the effect was inversely related to cell density of the SMCs. This stimulatory effect of lysates was completely blocked by antibody to PDGF. After the growth-promoting activity of lysates was eliminated by anti-PDGF, growth-inhibiting activity was revealed. Lysates prepared from old SMCs had significantly less capacity to inhibit target cell growth. In the presence of exogenous heparin both the serum- or PDGF-stimulated proliferation and serum-free proliferation of old SMCs were decreased to the level of proliferation of young SMCs. These results suggest that the balance between growth-promoting and growth-inhibiting factors is altered in SMCs from old rats. This may contribute to the increased proliferative capacity of these cells in culture and may facilitate the development of atherosclerosis with age.
Recombinant human interleukin 5 is a selective activator of human eosinophil function.
Authors: Lopez A F , Sanderson C J , Gamble J R , Campbell H D , Young I G , Vadas M A
Published: June 2004

Human rIL-5 was found to selectively stimulate morphological changes and the function of human eosinophils. This molecule is thus a prime candidate for the selective eosinophilia and eosinophil activation seen in disease.
Single cell origin of bigenotypic and biphenotypic B cell proliferations in human follicular lymphomas.
Authors: Cleary M L , Galili N , Trela M , Levy R , Sklar J
Published: June 2004

To investigate the possible relatedness of the subpopulations that make up so-called biclonal lymphomas, we examined five bigenotypic and biphenotypic follicular lymphomas using DNA probes specific for the t(14;18) chromosomal translocation, which is a characteristic feature of these neoplasms. On Southern blot analysis, both subpopulations from four of five lymphomas contained comigrating t(14;18) DNA rearrangements, confirming the single cell origins for these neoplasms. No comigrating t(14;18) DNA rearrangements were observed in the fifth lymphoma, but nucleotide sequence analysis of cloned, breakpoint DNA showed identical t(14;18) crossovers in the two subpopulations. The migration differences of both the Ig and chromosome 18 DNA rearrangements were shown to result from somatically acquired mutations of the Ig genes from the fifth lymphoma. These studies indicate that Ig gene rearrangements and idiotope expression are not consistently stable clonal markers since they are subject to variability as a result of somatic mutation. Although translocated chromosome 18 DNA rearrangements are more reliable, they may also vary among cells of some tumors since somatic mutation can affect, as well, DNA of translocated alleles in follicular lymphomas.
Calcium ionophore synergizes with bacterial lipopolysaccharides in activating macrophage arachidonic acid metabolism.
Authors: Aderem A A , Cohn Z A
Published: June 2004

LPS, a major component of Gram-negative bacterial cell walls, prime macrophages for greatly enhanced arachidonic acid [20:4] metabolism when the cells are subsequently stimulated. The LPS-primed macrophage has been used as a model system in which to study the role of Ca2+ in the regulation of 20:4 metabolism. The Ca2+ ionophore A23187 (0.1 microM) triggered the rapid release of 20:4 metabolites from LPS-primed macrophages but not from cells not previously exposed to LPS. Macrophages required exposure to LPS for at least 40 min before A23187 became effective as a trigger. A23187 (0.1 microM) also synergized with PMA in activating macrophage 20:4 metabolism. The PMA effect could be distinguished from that of LPS since no preincubation with PMA was required. A23187 greatly increased the amount of lipoxygenase products secreted from LPS-primed macrophages, leukotriene C4 synthesis being increased 150-fold. LPS-primed macrophages, partially permeabilized to Ca2+ with A23187, were used to titrate the Ca2+ concentration dependence of the cyclooxygenase and lipoxygenase pathways. Cyclooxygenase metabolites were detected at an order of magnitude lower Ca2+ concentration than were lipoxygenase products. The data suggest that Ca2+ regulates macrophage 20:4 metabolism at two distinct steps: an increase in intracellular Ca2+ regulates the triggering signal and relatively higher Ca2+ concentrations are required for 5-lipoxygenase activity.
Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone.
Authors: Austyn J M , Kupiec-Weglinski J W , Hankins D F , Morris P J
Published: June 2004

Using quantitative techniques we have shown elsewhere that dendritic cells (DC) migrate from blood into the spleen, under the control of T cells. Here we traced the localization of DC within the spleen and sought to explain the means by which they entered. DC were labeled with a fluorochrome, Hoescht 33342, and injected intravenously. Spleens were removed 3 or 24 h later and DC were visualized within particular areas that were defined by mAbs and FITC anti-Igs. At 3 h most DC were in the red pulp, whereas by 24 h the majority had homed to T-dependent areas of the white pulp and may have become interdigitating cells. Lymphoid DC, isolated from spleen and perhaps normally present in blood, may thus be a migratory stage distinct from the relatively fixed interdigitating cells. We also developed a frozen section assay to investigate the interaction of DC with various lymphoid elements. When DC were incubated on sections of spleen, at 37 degrees C but not at 4 degrees C they attached specifically within the marginal zone and did not bind to T areas; in contrast, macrophages attached only to red pulp and T cells did not bind specifically. However, DC did not bind to sections of mesenteric lymph node, whereas T cells localized in particular regions at 4 degrees C but not at 37 degrees C, probably the high endothelial venules. DC may thus express "homing receptors," similar to those of T cells, for certain endothelia. We propose that T cells can modify the vascular endothelium in certain areas to allow egress of DC from the bloodstream.
Chromosomal translocation involving the beta T cell receptor gene in acute leukemia.
Authors: Cleary M L , Mellentin J D , Spies J , Smith S D
Published: June 2004

DNA spanning a t(7;19) chromosomal translocation breakpoint was isolated from the human T cell line SUP-T7 established from an acute lymphoblastic leukemia. Nucleotide sequence analysis showed that the point of crossover on chromosome 7 occurred immediately adjacent to joining segment J beta 1.1 within the TCR-beta gene, suggesting that this translocation resulted from an error in TCR gene rearrangement. On chromosome 19, the translocation occurred within a previously uncharacterized transcriptional unit for which we propose the name lyl-1. An approximately 1.5-kb RNA is transcribed from this gene in a wide variety of hematolymphoid cell lines. The t(7;19) results in truncation of the lyl-1 gene and production of abnormal-sized RNAs, suggesting a role for lyl-1 in the pathogenesis of this leukemia.
Kirsten murine sarcoma virus abolishes interferon gamma-induced class II but not class I major histocompatibility antigen expression in a murine fibroblast line.
Authors: Maudsley D J , Morris A G
Published: June 2004

The effect of infecting fibroblasts with Kirsten murine sarcoma virus/murine leukemia virus (Ki-MSV/MLV) on constitutive and IFN-gamma-induced H-2 antigen expression was investigated. The fibroblasts used were two established cell lines (C3H10T1/2 and BALB/c3T3) and fresh embryo fibroblasts from C3H mice. Class I antigens were expressed constitutively by BALB/c3T3; infection with MLV, MSV or the two together had little effect on this constitutive expression. Class I antigens (H-2K, H-2D) were strongly induced on all three types of fibroblast by rIFN-gamma, and infection had little effect on this. None of the fibroblasts expressed constitutively detectable levels of class II antigen; however, C3H10T1/2 fibroblasts could be induced for both H-2A and H-2E by IFN-gamma. Infection of C3H10T1/2 with helper-free Ki-MSV, or MSV together with MLV, completely abolished this induction of class II antigens, while infection with MLV alone had little effect, implying that the abolition of class II induction was due to genomic regions of Ki-MSV not shared with Ki-MLV, probably the v-Ki-ras gene.
A monoclonal antibody (3G5)-defined ganglioside antigen is expressed on the cell surface of microvascular pericytes.
Authors: Nayak R C , Berman A B , George K L , Eisenbarth G S , King G L
Published: June 2004

The identification of microvascular pericytes in vitro relies principally on morphological characteristics and growth dynamics, as there is a paucity of immunochemical markers for these cells. Consequently, an attempt was made to identify mAb reagents that would aid in both the rapid identification and enrichment of retinal capillary pericytes in vascular cell cultures. A panel of mAbs raised by xenogeneic immunization of mice with various tissues was screened for immunoreactivity with dissociated cultures of bovine retinal capillary pericytes. Two antibodies from the panel (3G5 and HISL-8) were seen to react with pericytes by indirect immunofluorescence. The mAb 3G5 was selected for further study. mAb 3G5 did not react with dissociated cultures of smooth muscle cells, endothelial cells, or retinal pigmented endothelial cells. The pericyte 3G5 antigen was insensitive to the action of trypsin; therefore, mAb 3G5 was used to selectively purify pericytes from trypsinized mixed retinal cell cultures by flow cytometry. 3G5+ pericytes (representing 8% of cells in a mixed retinal cell culture) were enriched at least nine-fold to represent greater than 70% of cells. The mAb 3G5 stained retinal capillaries in vivo with a fluorescence distribution consistent with pericyte staining. The 3G5 antigen of cultured pericytes was found to be a glycolipid of mobility intermediate between ganglioside markers GM1 and GM2.
The antitumor function of tumor necrosis factor (TNF), I. Therapeutic action of TNF against an established murine sarcoma is indirect, immunologically dependent, and limited by severe toxicity.
Authors: Havell E A , Fiers W , North R J
Published: June 2004

The ability of murine recombinant tumor necrosis factor (rTNF) and natural TNF in tumor-necrotizing serum (TNS) to cause regression of the SA1 sarcoma was investigated. We found that to cause regression of a 9-d SA1 sarcoma, near lethal quantities of rTNF and TNS had to be given to the host. However, even at these highly toxic doses, rTNF was not reliable at causing complete tumor regression. On the other hand, both types of TNF were reliable at causing a tumor hemorrhagic reaction that resulted in the destruction of greater than 75% of the tumor's center in 24 h. The TNF-induced hemorrhagic reaction involved the development of numerous petechial hemorrhages in the tumor's vascular bed, which apparently resulted from destruction of the tumor's blood vessels. It was possible to follow the development of the hemorrhagic reaction against time after giving rTNF or TNS by measuring the intratumor extravasation of 51Cr-labeled syngeneic red cells. According to this method, TNF-induced intratumor hemorrhaging was in progress within 1 h of giving TNF and continued for about a 6-h period. However, the hemorrhagic reaction was greatly reduced and complete regression of the rim of the living tumor tissue that survived hemorrhagic necrosis failed to occur, if SA1 sarcoma was growing in T cell-deficient (TXB) mice. This indicates that the TNF-induced hemorrhagic reaction is partly dependent, and the tumor regression that follows is completely dependent on host immunocompetence. This suggests in turn, that rTNF does not directly destroy SA1 tumor cells in vivo, even though it was shown that it can destroy SA1 tumor cells in vitro. This interpretation is supported by the additional findings that rTNF was no more therapeutic against a 3-d (3-mm) SA1 than against a 9-d (8-mm) SA1, and was no more therapeutic when injected directly into the tumor than when injected intravenously. Lastly it was possible to completely inhibit the ability of rTNF and TNS to cause tumor hemorrhagic necrosis and regression by infusing the host with a monospecific, polyvalent anti-rTNF antibody that neutralized the cytotoxic action of rTNF in vitro.
A high proportion of T lymphocytes that infiltrate H-2-incompatible heart allografts in vivo express genes encoding cytotoxic cell-specific serine proteases, but do not express the MEL-14-defined lymph node homing receptor.
Authors: Mueller C , Gershenfeld H K , Lobe C G , Okada C Y , Bleackley R C , Weissman I L
Published: June 2004

The role of cytotoxic cells in in vivo immune functions such as allograft rejection is unknown. To begin to assess the function of cytolytic cells in vivo we have begun with cytolytic cell-specific functional molecules: we have isolated and characterized cytolytic cell-specific cDNA clones from cytolytic T cell clones, both encoding distinct serine esterases. The HF gene encodes a trypsin-like enzyme while the C11 gene encodes an enzyme with likely specificity for acidic residues. Here we demonstrate, using in situ hybridization with RNA probe, that both genes are expressed selectively in a subset of T lymphocytes that have infiltrated cardiac allografts. The phenotype of these cells is consistent with the most frequent phenotype of active CTL raised in vitro: they are predominantly CD4-, CD8+, MEL-14- T cell blasts. Thus the expression of these genes, each of which encodes serine esterase found in killer cell granules in vitro, is a valid marker for these cells in vivo as well. The kinetics of their accumulation is consistent with, but not proof of, a putative role in allograft rejection. It is likely that HF and C11 gene expression will be of diagnostic value.
Expression and characterization of a truncated murine Fc gamma receptor.
Authors: Qu Z X , Odin J , Glass J D , Unkeless J C
Published: June 2004

We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.
Cachectin/tumor necrosis factor induces cachexia, anemia, and inflammation.
Authors: Tracey K J , Wei H , Manogue K R , Fong Y , Hesse D G , Nguyen H T , Kuo G C , Beutler B , Cotran R S , Cerami A
Published: June 2004

Cachexia is a potentially lethal syndrome of unknown etiology characterized by anorexia, weight loss, and protein wasting that frequently complicates the treatment of chronic inflammation and cancer. Cachectin/TNF was isolated during the search for a humoral mediator of cachexia and found to stimulate the breakdown of energy stores from adipocytes and myocytes in vitro, but the chronic effects of the monokine in vivo are not known. Sublethal doses of recombinant human cachectin administered twice daily for 7-10 d caused cachexia in rats, as evidenced by reduced food intake, weight loss, and depletion of whole-body lipid and protein stores. Significant anemia is also observed and found to be the result of decreased red blood cell mass, not expanded plasma volume. Leukocytosis and histopathological evidence of tissue injury and inflammation are observed in several organs, including omentum, liver, spleen, and heart. These data suggests that the exposure of the normal host to cachectin is capable of inducing a pathophysiological syndrome of cachexia, anemia, and inflammation similar to that observed during inflammatory states or malignancy.
Population kinetics of rat peripheral B cells.
Authors: Gray D
Published: June 2004

Currently available estimates of B cell life span vary from 4 d to 6 wk. The discrepancy may have arisen out of the selective effects of stress and drug cytotoxicity on short-lived populations. In this report, bromodeoxyuridine (BUdR), a drug that incorporates into the DNA of dividing cells, has been fed to rats in their drinking water, eliminating stressful injection procedures. Labeled cells in the recirculating B cell pool are identified in tissue sections using an mAb to BUdR. BUdR is shown to have no cytostatic effects at the dose used. Over a 5-d period of infusion, only 20% of the peripheral recirculating pool incorporate label (approximately 4% per day); labeling over various periods indicates that the peripheral B cell pool turns over in approximately 4 wk. To distinguish between turnover due to incorporation of new B cells into the peripheral pool and division of antigen-activated B cells rats underwent two consecutive periods of labeling, first with [3H]thymidine for 5 d and then with BUdR for a further 5 d. Virgin B cells newly derived from dividing precursors in the bone marrow do not continue to proliferate in the periphery, while activated cells undergo several rounds of division during both labeling periods. The results indicate that 3-4% of the peripheral pool is replaced by new B cells each day, while 0.3-0.6% become part of activated clones every day. Assuming that the peripheral pool of the rat contains 10(9) B cells, then 3-4 X 10(7) new B cells become stably incorporated per day. This represents approximately 10% of the putative output of the bone marrow.
Identification of homologous regions in human immunodeficiency virus I gp41 and human MHC class II beta 1 domain. I. Monoclonal antibodies against the gp41-derived peptide and patients' sera react with native HLA class II antigens, suggesting a role for autoimmunity in the pathogenesis of acquired immune deficiency syndrome.
Authors: Golding H , Robey F A , Gates F T , Linder W , Beining P R , Hoffman T , Golding B
Published: June 2004

Homologous regions of five amino acids each, were identified in the NH2-terminal domain of human class II beta chains and the COOH terminus of HIV I envelope protein. The homologous regions are highly conserved among different DR and DQ alleles and also among different isolates of HIV. Septamers containing these sequences were synthesized and used for the generation of murine mAbs. The mAbs selected for this study were raised against the HIV I-derived peptide and reacted strongly not only with the immunizing peptide, but also with the homologous class II-derived peptide. These mAbs also reacted with native MHC class II antigens expressed on human B cell lines and on murine fibroblast L cell lines transfected with the genes coding for the alpha and beta chains of human class II antigens. Furthermore, sera from 36% of AIDS patients tested contained antibodies that reacted with the class II-derived peptide, as well as with intact class II molecule-rich cell extracts. Such antibodies in HIV I-infected individuals may recognize self class II antigens, triggering autoimmune mechanisms that could contribute to the development of immunodeficiency in AIDS patients.
Induction of high-affinity interleukin 1 receptor on human peripheral blood lymphocytes by glucocorticoid hormones.
Authors: Akahoshi T , Oppenheim J J , Matsushima K
Published: June 2004

The in vitro effect of glucocorticoids (GCs) on IL-1-R expression of human PBMCs was investigated. Both physiological and pharmacological concentration ranges of GC increased the specific binding of 125I-labeled human rIL-1 alpha to PBMCs. This enhancement was specific for GC, since other steroid hormones, such as progesterone, 17 beta-estradiol, and testosterone failed to elevate the binding of 125I-IL-1 alpha to PBMCs. The effect was time dependent with maximal effect occurring 6 h after treatment and dose dependent with half-maximal effect elicited by 100 nM prednisolone. Scatchard plot analysis indicated that 125I-IL-1 alpha binding increased from approximately 100 IL-1-R per cell to 2 X 10(3) receptors per cell without a major change in affinity (Kd = 2.6 X 10(-10) M). The subpopulation of PBMCs induced by GC to express higher levels of IL-1-R consisted predominantly of B lymphocytes, but not T lymphocytes, large granular lymphocytes, or monocytes. GCs also induced the expression of IL-1-R on some other cell types, including normal human dermal fibroblasts and the human large granular lymphocyte cell line YT. Since cycloheximide and actinomycin D inhibited the induction of IL-1-R by GC, synthesis of both new RNA and protein seems to be required for IL-1-R induction. This study presents the first evidence of upregulation of the receptors for IL-1 by GC, and may account for the reported enhancement of in vitro and in vivo humoral immune responses by GCs.
In vivo incisional wound healing augmented by platelet-derived growth factor and recombinant c-sis gene homodimeric proteins.
Authors: Pierce G F , Mustoe T A , Senior R M , Reed J , Griffin G L , Thomason A , Deuel T F
Published: June 2004

Human platelet-derived growth factor (hPDGF) is likely to be important in stimulating tissue repair, based upon its in vivo chemotactic and stimulatory activities for inflammatory cells and fibroblasts and upon the presence of PDGF and related proteins in platelets, macrophages, and activated fibroblasts, cell types that make up the milieu of the healing wound. Recombinant human c-sis (rPDGF-B), homodimers of the B chain of PDGF, were compared with hPDGF in vitro. rPDGF-B was immunologically similar to hPDGF and, at identical concentrations, similar to hPDGF in stimulating fibroblast mitogenesis and chemotaxis of polymorphonuclear leukocytes, monocytes, and fibroblasts. Purified hPDGF and rPDGF-B were also tested in vivo for potency in a model of tissue repair using a linear incision wound through rat dermis. A single application of hPDGF or rPDGF-B (2-20 micrograms/wound) in a slow release vehicle at the time of wounding resulted in a dose-dependent, statistically highly significant increase of breaking strength of treated wounds. Wound healing in animals treated with rPDGF-B was 170% stronger and accelerated by 2 d during the first week over control wounds and by 4-6 d over the next 2 wk. Histologic evaluation of growth factor-treated wounds correlated the in vitro chemotactic activity and the accelerated healing of wounds with a striking inflammatory cell infiltrate early after wounding, markedly increased formation of granulation tissue by 4-d, and increased fibrosis by 14 d in comparison to control wounds. The results thus demonstrate that rPDGF-B is fully active in in vitro tests of mitogenesis and chemotaxis and, for the first time, demonstrate directly that PDGF significantly advances wound healing in incisional wounds of experimental animals.
Heterogeneity of helper/inducer T lymphocytes. II. Effects of interleukin 4- and interleukin 2-producing T cell clones on resting B lymphocytes.
Authors: Boom W H , Liano D , Abbas A K
Published: June 2004

To compare the helper function of murine T cell clones that secrete IL-2 and IFN-gamma (Th1 cells) or IL-4 and IL-5 (Th2), purified resting B cells were stimulated with F(ab')2 rabbit anti-mouse Ig (RAMG) and rabbit Ig-specific, class II MHC-restricted cloned T cells belonging to the two subsets. Both Th2 clones examined induced strong proliferative responses of B cells in the presence of RAMG, as well as the secretion of IgM and IgG1 antibodies. In contrast, the Th1 clones tested failed to stimulate B cell growth or antibody secretion. Th2-mediated B cell activation was dependent on IL-4 and IL-5, and was also inhibited by IFN-gamma or IFN-gamma produced by Th1 cells present in the same cultures. However, the failure of Th1 cells to help resting B cells could not be reversed with neutralizing anti-IFN-gamma antibody. In addition to this inhibitory effect, IFN-gamma was required for the secretion of IgG2a antibody, particularly when B cells were stimulated with polyclonal activators such as LPS. Finally, both sets of T cell clones secreted lymphokines when stimulated with purified B cells and RAMG. These experiments demonstrate that T cells that differ in lymphokine production also differ in their ability to help B cells as a result of cognate interactions at low concentrations of antigens. Moreover, IL-4, IL-5, and IFN-gamma serve different roles in the T cell-dependent proliferative and differentiative responses of resting B lymphocytes.
Recombinant interleukin 2 or 5, but not 3 or 4, induces maturation of resting mouse B lymphocytes and propagates proliferation of activated B cell blasts.
Authors: Karasuyama H , Rolink A , Melchers F
Published: June 2004

Plasmacytoma transformants of the X63-Ag8-653 cell line carrying an expression vector with either IL-2, -3, -4, or -5 cDNA were established that secrete the corresponding ILs at high rates. The four mouse ILs (mILs) were then tested as single ILs and in combinations for their effects on the maturation of resting and proliferation of activated normal mouse splenic B cells. mIL-3 and mIL-4 were inactive in all assays. mIL-2, as well as mIL-5, synergized with Ig-specific antibodies and B cell growth factor alpha (BCGF-alpha) to stimulate successive rounds of B cell division with LPS-activated B cells. This activity as BCGF-beta was effective at concentrations similar to those at which mIL-2 induced proliferation of the CTL-L T cell line, indicating a high-affinity interaction of both mIL-2 and mIL-5 with their corresponding receptors on activated B cells. mIL-5 and maybe IL-2 also induced maturation of resting B cells to Ig-secreting cells without proliferation. This B cell maturation factor (BMF) activity of mIL-5 was as effective as its BCGF-beta activity, while the BMF activity of mIL-2 was at least 10(2)-fold less effective. BMF activity of mIL-2, but not mIL-5, was blocked by anti-Il-2-R antibodies, indicating that mIL-2 and mIL-5 use separate receptors for B cell signaling. mIL-2, as well as mIL-5, furthermore, acted as filler activities when proliferation in the presence of Ig-specific antibodies and BCGF-alpha was measured with as little as 500 B cells. In the case of mIL-5, this was also true for maturation of that few cells. Limiting dilution analyses showed that approximately 1-2% of the resting B cells matured without division, while 30-100-fold fewer cells (0.03-0.06%) proliferated and matured in response to IL-5. A single IL, therefore, is capable of inducing maturation and of stimulating mitotic cell cycle progression of normal B cells.