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Biomedical subjects

M Hagen

Publications and source records attributed to M Hagen.

At least 37 records · Page 2Linked to original sources
PubMed1995

The Fc epsilon RII/CD23 gene is actively transcribed during all stages of murine B-lymphocyte development.

It is generally accepted that the expression of Fc epsilon RII/CD23 on the surface of the B-lineage cells is restricted to the stage of the resting, mature (sIgM+/sIgD+) B-lymphocyte. However, it is unknown whether activation of the Fc epsilon RII/CD23 gene is also restricted to the stage of the mature B-lymphocyte. To address this question we investigated a panel of B-lineage cell lines for the presence of transcripts encoding Fc epsilon RII/CD23. We detected transcripts in 16 of 26 B-lineage cell lines representing the entire spectrum of B-cell development. In most cases (13 of 16) active transcription of the murine Fc epsilon RII/CD23 gene was not coupled with the expression of cell surface Fc epsilon RII/CD23 expression did not hold for all murine B-cell lines. One post-switch B-cell line (sIgM-/sIgG+) expressed Fc epsilon RII/CD23 on the cell surface and another could be induced with IL-4 and LPS to express surface Fc epsilon RII/CD23. Transcription of the murine CD23 gene in the absence of cell surface expression of Fc epsilon RII/CD23 does not appear to simply be an aberrant feature of transformed B-cells since we found transcripts, but not surface expression, in some normal splenic and peritoneal B-lymphocytes. Our findings suggest that the potential for expression of Fc epsilon RII/CD23 may occur over a much broader development window of the B-lineage than previously suspected. Transcription of the Fc epsilon RII/CD23 gene, in the absence of detectable cell surface protein expression in B-lineage cell lines, and in sort-purified B-lymphocyte subpopulations, implies that in addition to regulatory mechanisms already known, murine CD23 is also regulated through post-transcriptional mechanisms that have not yet been characterized.

Animals↗
PubMed1995

Functional consequences of the interaction between T-cell antigen receptors and Fc gamma Rs on T cells.

Two series of experiments are presented indicating that Fc gamma II receptors can interfere with the antigen receptor-induced signaling on T cells. It has been previously described that a 13 amino acid motif on the cytoplasmic portion of Fc gamma II beta 1 can abrogate the antigen receptor-initiated signals mediated through consensus motifs present on the cytoplasmic portion of Ig alpha and Ig beta chains. Similar activating motifs are crucial to T-cell receptor (TCR) signaling. A splenic gamma/delta T-cell hybridoma that expressed the Fc gamma RII beta 1 receptor helped to establish that this receptor can also interfere with TCR-induced activation. The cytoplasmic portion of human Fc gamma RIIa has an activation motif similar to the activation motifs present on the TCR. Using a transgenic mouse in which the T cells express the human Fc gamma RIIa transgene, we demonstrated that despite the common activation motif, the TCR and human Fc gamma RIIa-induced signals are different. Additionally, the human Fc gamma RIIa expressing T cells exhibit an enhanced TCR response both in vitro and in vivo.

Antigens, CD↗
PubMed1995

Developmental regulation of Fc epsilon RII/CD23 expression in B-lineage cells: evidence for transcriptional and post-transcriptional levels of control.

The present studies show that the expression of cell surface Fc epsilon RII/CD23, detected with the monoclonal anti-Fc epsilon RII/CD23 antibody B3B4 or by the binding of IgE, is not restricted to the stage of resting mature virgin B lymphocytes. Murine CD23 was detected as a cell surface protein on two sIgG+ B-cell lines. Moreover, we detected full-length transcripts for Fc epsilon RII/CD23 in several members of a panel murine B lymphoid lineage cell lines representative of all stages of murine B lymphocyte development. Our findings suggest that regulation of CD23 translation may differ between B-cell lines at various stages of differentiation. The detection of mRNA transcripts for Fc epsilon RII/CD23 was not restricted to transformed cell lines. Fc epsilon RII/CD23 transcripts were amplified by RT-PCR from peritoneal and splenic B lymphocyte subpopulations that were sorted by flow cytometry into populations that did not express surface Fc epsilon RII/CD23. Our findings suggest that Fc epsilon RII/CD23 transcription and translation are not necessarily restricted to the narrow developmental window of murine B lymphocyte differentiation as previously thought. Our findings imply that Fc epsilon RII/CD23 may be expressed at the protein level at various stages of murine B lymphocyte differentiation. Investigations into the expression patterns and potential mechanisms of regulation of Fc epsilon RII/CD23 could provide insight into the basis for the wide range of immunological functions that have been proposed for Fc epsilon RII/CD23.

Animals↗
PubMed1995

Functional significance of CD23- on CD23-transfected Th2 clone.

CD23, a low-affinity IgE Fc receptor, is not displayed on most resting T cells but its expression has been shown to be transiently induced in vivo and in vitro on some CD4+ T cells [1-4] and in vivo on CD8+ T cells by IgE-secreting hybridoma tumors [5]. To investigate the functional role of CD23 on T cells, we inserted a CD23 construct into an expression vector driven by a CD2 promoter and transfected it into a murine Th2 clone D10.G4.1 (D10). We stimulated the transfected D10 cells (D10.3M.24) with anti-TCR antibody in the presence or absence of IgE, and measured IL-4, IL-5 and IL-6 production in the culture supernatants. Activation of D10.3M.24 cells by anti-TCR antibody induced greater levels of IL-4, IL-5 and IL-6 production, when the TCR and CD23 were co-crosslinked by TNP anti-TCR and IgE anti-TNP antibodies. IgG anti-TNP antibody did not enhance lymphokine production by D10.3M.24 cells. The enhanced lymphokine production by IgE was blocked by monoclonal anti-CD23 antibody. IgE anti-TNP antibody did not enhance lymphokine production by the wild-type D10 cells induced by TNP anti-TCR antibody. These studies show that when co-crosslinked with the TCR, CD23 can modulate the lymphokine production in activated Th2 cells. Since CD23 binds to IgE and also binds to CD21 [6], a complement receptor commonly expressed on B cells, T-cell CD23 could play an immunoregulatory role during cognate T-B cell interaction and during IgE antibody responses.

Animals↗
PubMed1995

Potential role of Fc gamma R in early development of murine lymphoid cells: evidence for functional interaction between Fc gamma R on pre-thymocytes and an alternative, non-Ig ligand on thymic stromal cells.

During early development of the murine fetal thymus a fraction of Thy+ thymocytes express Fc gamma receptors. Concurrently, a small fraction of Thy- CD44- thymocytes bind recombinant soluble Fc gamma R. These findings suggested the possibility that Fc gamma R+ pre-T cells interact with a non-Ig ligand present on fetal thymic stromal cells. Evidence in support of this concept was the finding that experimental manipulation of this putative receptor-ligand pair by anti-receptor antibody or recombinant soluble Fc gamma R influenced the developmental pattern of alpha/beta-TCR+ T cells. The restricted expression of CD16 on some pre-B cells raises the possibility that an Fc gamma R-dependent step may play a similar role in B-cell development.

Animals↗
PubMed1995

Human fetal adrenal hydroxysteroid sulphotransferase: cDNA cloning, stable expression in V79 cells and functional characterisation of the expressed enzyme.

Dehydroepiandrosterone sulphate (DHEAS) is a major adrenal secretory product, particularly in the fetus where it serves as a substrate for oestrogen biosynthesis by the placenta. The enzyme in the adrenal responsible for synthesising DHEAS, hydroxysteroid sulphotransferase (HST), is therefore essential for human development. We have isolated a full-length cDNA clone, encoding human fetal adrenal HST, and constructed a stable cell line expressing it by transfection into V79 Chinese hamster lung fibroblast cells. This cDNA was essentially identical to that isolated from adult human liver, where the role of HST is less well understood. This recombinant cell line allowed determination of the substrate specificity and kinetic properties of this enzyme towards various steroid hormones, and by comparison of these activities with human liver cytosol we have shown that HST is the major sulphotransferase responsible for the sulphation of DHEA, androsterone and pregnenolone in man and that, functionally, the hepatic and adrenal enzymes are very similar. The expressed HST was also active with testosterone, cortisol (although at low levels) and the xenobiotic 17 alpha-ethinyloestradiol, but not with oestrone or 1-naphthol. We have therefore created a valuable resource for the study of this important enzyme.

Adrenal Glands↗
PubMed1995

Routine tests during follow-up of patients after primary treatment for operable breast cancer. International (Ludwig) Breast Cancer Study Group (IBCSG)

BACKGROUND: Follow-up tests for patients after diagnosis and primary treatment of breast cancer are routinely performed. However, the usefulness of these follow-up parameters remains unclear. We determined the yield of a variety of blood tests used to detect the presence of overt metastatic disease. METHODS: 4105 patients enrolled in International (Ludwig) Breast Cancer Study Group (IBCSG) randomized clinical trials from 1978 to 1985 were analyzed for abnormal or equivocal findings in six routine blood tests obtained every 3 months for the first two years, every six months for years 3-5 and yearly thereafter. The relationship of test results to the occurrence of overt metastatic disease was evaluated. The relapses were categorized as follows in order to estimate the yield of the different tests for relevant sites of metastases: any breast cancer relapse, bone +/- other; liver +/- other. RESULTS: Alkaline phosphatase alone was abnormal in a high proportion of patients with either bone metastases, liver metastases, or both. SGOT and gamma-GT were also sensitive for patients with liver metastases. Bilirubin, serum calcium, and serum creatinine were relatively insensitive indicators of relapse. Abnormal test results were reported sometime during a patient's disease-free period for 3% to 6% of patients, depending on the test. CONCLUSIONS: Alkaline phosphatase was the most effective blood test to distinguish patients with relapse from those without relapse. It is inexpensive and its yield is relatively high for predicting liver and bone metastases. The routine use of the other tests analyzed to detect metastases was not justified.

Breast Neoplasms↗
PubMed1995

The role of template-primer interactions in cleavage and initiation by the influenza virus polymerase.

An in vitro cleavage/initiation assay was used to analyse cleavage site choice and transcription initiation by the influenza virus polymerase. A synthetic mRNA which is cleaved by the polymerase to produce a single 11 base primer fragment was altered around this cleavage site. Depending upon the mutations made, alternative cleavage sites were used. This system was then used in extracts from recombinant vaccinia virus infected cells which express the polymerase. These extracts require the addition of a synthetic vRNA in order to induce cleavage and initiation activity. The data show that the choice of cleavage site is wholely controlled by the mRNA and does not depend upon interactions with the vRNA template. However, the site of initiation of the cleaved primer on the template is influenced by template-primer interactions.

Base Sequence↗
PubMed1994

B lymphocytes of mice display an aberrant activation phenotype and are cell cycle arrested in G0/G1A during acute infection with Trypanosoma brucei.

Humans and domestic animals with African trypanosomiasis exhibit abnormalities of immune function characterized by polyclonal lymphocyte activation and, paradoxically, progressive immunodeficiency. Mice infected with Trypanosoma brucei clone IaTat1.2 develop a fulminant parasitemia by day 5 of infection. B lymphocytes isolated from spleens of infected mice display an aberrant activation phenotype manifested by decreased CD23 and surface IgM, increased CD69 and L-selectin, and normal levels of surface IgD, MHC class II, CD32, and transferrin receptor. Kinetic analyses showed that CD23 and surface IgM were continuously down-regulated from day 2 through day 5, whereas MHC class II was elevated on days 2 and 3, but returned to normal levels by day 5, suggesting that CD23 and MHC class II are independently regulated in T. brucei infection. The aberrant activation phenotype of B cells responding in vivo to T. brucei was accompanied by impaired responsiveness of these B cells to mitogenic stimulation in vitro. The pathologic phenotypic and functional properties of B lymphocytes from T. brucei-infected mice can be partially accounted for by the virtual total arrest of B cells in G0/G1A of the cell cycle. The B lymphocyte alterations observed in the present studies provide new insight into the immunopathology of African trypanosomiasis. We propose that terminal infection with T. brucei induces early activation events in host B lymphocytes, but that the activation response becomes aberrant by the development of what seems to be a total block in cell cycle progression.

Animals↗
PubMed1994

Biochemical studies on capped RNA primers identify a class of oligonucleotide inhibitors of the influenza virus RNA polymerase.

A synthetic 67-nt RNA substrate, containing a 32P-labeled cap-1 structure (m7G32pppGm) was specifically cleaved by the influenza virus RNA polymerase (EC 2.7.7.48) to yield a single capped 11-nt fragment capable of directly priming transcription. An analysis of systematic truncations of this RNA substrate demonstrated that an additional nucleotide beyond this cleavage site was required for cleavage. The minimal RNA chain length required for priming activity was found to be 9 nt, while in contrast an RNA chain length of at least 4 nt was required for efficient binding to the viral polymerase. On the basis of these chain length requirements we show that a pool of capped oligonucleotides too short to prime transcription, but long enough to bind with high affinity to the viral polymerase, are potent inhibitors of cap-dependent transcription in vitro.

Base Sequence↗
PubMed1994

Methods for studying Fc receptor expression.

In the past two decades, rapid progress has been made in the field of Fc receptors. One of the reasons for this development has been the advances and improvements in the methodologies used to detect and analyze Fc receptors. This review describes the methods used to detect the murine and human Fc receptors of the various immunoglobulin isotypes at the RNA, protein, and functional level. We have given special attention to the binding assays used to detect Fc receptors through immunoglobulins or anti-Fc receptor monoclonal antibodies as probes. The various Fc receptor types exhibit a marked heterogeneity and we present a list of reagents that can differentiate between the different Fc receptor forms. These methods can detect Fc receptors on the cell membrane, as well as the cytoplasmic and secreted forms. Recent advances in the use of PCR and RNA/DNA analyses to study Fc receptor detection, expression, and function are also reviewed. The advantages and drawbacks of the various experimental approaches are presented. In addition, an appendix provides detailed protocols for the detection of Fc receptors by confocal immunofluorescence microscopy, rosetting, cytofluorometry, immunoprecipitation, and PCR.

Animals↗
PubMed1994

Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease activity.

Influenza virus polymerase complexes that were expressed in the absence of genomic viral RNA and nucleoprotein were examined for endonuclease activity and transcriptase ability in vitro. Nuclear extracts of cells that express influenza virus polymerase through recombinant vaccinia virus infection did not display specific endonuclease activity in vitro. This polymerase presumably represents an early form of enzyme present in infected cells prior to ribonucleoprotein assembly. Upon addition of a virus-like model RNA template, containing the partially complementary sequence found at the ends of viral RNA, endonuclease activity is stimulated in a concentration-dependent and sequence-specific manner. Once stimulated, the polymerase is able to elongate from the added viral template. Thus, addition of viral template is required for polymerase activity, while the presence of nucleoprotein is not required for limited transcription. Also, full activation of this recombinant viral polymerase is dependent on the presence of both the 3' and 5' ends of the viral genome, as model RNA containing either end alone could not effectively trigger the endonuclease.

Base Sequence↗
PubMed1994

Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs.

The enzymatic activity of recombinant influenza virus RNA polymerase is strictly dependent on the addition of a template RNA containing 5' and 3' viral sequences. Here we report the analysis of the binding specificity and physical characterization of the complex by using gel shift, modification interference, and density gradient techniques. The 13S complex binds specifically to short synthetic RNAs that mimic the partially double stranded panhandle structures found at the termini of both viral RNA and cRNA. The polymerase will also bind independently to the single-stranded 5' or 3' ends of viral RNA. It binds most strongly to specific sequences within the 5' end but is unable to bind these sequences in the context of a completely double stranded structure. Modification interference analysis identified the short sequence motifs at the 5' ends of the viral RNA and cRNA templates that are critical for binding.

Amino Acid Sequence↗
PubMed1992

Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f. To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli. The expressed protease underwent autoproteolytic processing at two independent sites. The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease. The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus. A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria. The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria. This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease. ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E. coli. This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus.

Blotting, Western↗
PubMed1992

Effect of anti-DNP IgG1- and IgG2a-secreting hybridomas in vivo on the development of an anti-DNP IgE antibody response in mice.

We reported previously that CBA mice pretreated with dinitrophenyl-Bordetella pertussis (DNP-BP) conjugates exhibited sharply decreased anti-DNP IgE, and increased IgG2a antibodies following immunization with DNP-ovalbumin (DNP-OA) in alum. The objective of the present experiments was to determine whether the decrease in anti-DNP IgE was attributed to a regulatory effect exerted by IgG2a antibodies. Anti-DNP monoclonal antibodies (Mab) of the IgG1 or IgG2a isotype were passively transferred to mice, 24 h before a primary immunization with DNP-OA in alum. Anti-DNP IgE production was drastically suppressed in recipients of IgG1 but not of IgG2a Mab. Similar results were obtained when the Mab were endogenously produced by intraperitoneal implantation of anti-DNP-secreting hybridomas into (BALB/cxCBA)F1 (BCF1) mice. However, neither IgG1 nor IgG2a isotypes suppressed IgE antibody production if the hybridoma implantation took place 10 days after hapten priming. These results are, to our knowledge, the first to show a clear dissociation between the effect of either passively transferred or endogenously secreted IgG1 and IgG2a antibodies in their ability to inhibit a primary anti-hapten IgE antibody response.

Animals↗