Search PubMed⌕ Search

Biomedical subjects

A Saxon

Publications and source records attributed to A Saxon.

At least 55 records · Page 3Linked to original sources
PubMed1995

B cell lines from a subset of patients with common variable immunodeficiency undergo enhanced apoptosis associated with an increased display of CD95 (Apo-1/fas), diminished CD38 expression, and decreased IgG and IgA production.

Investigation of common variable immunodeficiency (CVI) is hampered by lack of a suitable in vitro models. We have developed EBV-transformed B lymphoblastoid cell lines from a selected subset of CVI patients and characterized them for phenotypic and functional properties that provide evidence for their representation of the CVI disease state. B cell lines from the patients expressed increased levels of sIgM and reduced levels of sIgD and sIgG. Essentially none of the CVI-derived B cell lines produced IgG and IgA while all produced IgM, in contrast to normal B cell lines that produced large amounts of IgG and IgM and detectable levels of IgA. Expression of CD95 (fas/Apo-1), a molecule that can induce apoptosis, was increased on the CVI B cell lines while CD38, a novel signaling molecule whose stimulation may prevent apoptosis, showed reduced expression. The B cell lines from the CVI patients exhibit increased apoptosis in vitro spontaneously, in response to anti-CD95 mAb and to X-irradiation. These phenotypic and functional changes are similar to findings on freshly derived B cells from the patients. EBV-derived B cell lines from patients with hyper-IgM immunodeficiency and X-linked agammaglobulinemia did not demonstrate increased CD95 expression or enhanced apoptosis. Thus the EBV-derived B cell lines from our selected CVI patients manifest many characteristics of the patients' fresh cells and may provide critical reagents for the further elucidation of the nature of the B cell dysfunction in the selected subset of CVI patients.

ADP-ribosyl Cyclase↗
PubMed1995

The human immune system in hu-PBL-SCID mice.

Severe combined immunodeficiency (SCID) mice can be stably grafted with human peripheral blood lymphocytes, creating hu-PBL-SCID chimeras; essentially, these are mice with a human immune system. Here, Magdalena Tary-Lehmann, Andrew Saxon and Paul Lehmann discuss the immunobiology of these chimeras. The authors propose that hu-PBL-SCID chimerism evolves in two phases. During the first three weeks after grafting, many of the injected cells survive and the human immune system is functional. Subsequently, anti-mouse-reactive clones are selected and the immune system becomes nonfunctional. The implications of this scenario for the utilization of the hu-PBL-SCID model are discussed.

Animals↗
PubMed1995

Enhanced human IgE production results from exposure to the aromatic hydrocarbons from diesel exhaust: direct effects on B-cell IgE production.

Epidemiologic and experimental studies suggest that air pollution, and particularly diesel exhaust particles (DEPs) may play a role in the increasing prevalence and severity of airway allergic disease. We show that the extract of polyaromatic hydrocarbons (PAHs) from DEPs (PAH-DEP) enhances human IgE production from purified B cells. Interleukin-4 plus CD40 monoclonal antibody-stimulated IgE production was enhanced 20% to 360% by the addition of PAH-DEP over a period of 10 to 14 days. This effect was increased when PAH-DEP was added 2 to 5 days after cultures were initiated. PAH-DEP itself did not induce IgE production or synergize with interleukin-4 alone to induce IgE from purified B cells, suggesting that it was enhancing ongoing IgE production rather than inducing germline transcription or isotype switching. The prototype nonmetabolized aromatic hydrocarbon 2,3,7,8 tetracholorodibenzo-p-dioxin, which functions solely through activation of the cytosolic aromatic hydrocarbon receptor complex, also increased IgE production. Additionally, the pattern of mRNAs coding for distinct isoforms of the epsilon chain was altered by PAH-DEP, and B-cell expression of the low-affinity IgE receptor was upregulated by PAH-DEP. Enhanced IgE production in the human airway, resulting from exposure to PAH-DEP, may be an important factor in the increase in airway allergic disease.

Adult↗
PubMed1995

B cells from a distinct subset of patients with common variable immunodeficiency (CVID) have increased CD95 (Apo-1/fas), diminished CD38 expression, and undergo enhanced apoptosis.

We investigated the role of apoptosis in the differentiation failure of B cells from a selected subpopulation of patients with CVID delineated by B cell surface marker analysis, in vitro IgE response, and molecular markers of B cell VH gene repertoire. These patients had altered display of B cell surface molecules that play a role in apoptosis. The patients' B cells had a 4.5-250-fold increase in CD95 (Apo-1, fas) expression and increased CD95 display on their T cells. CD38, a molecule important in preventing germinal centre B cell apoptosis, was reduced on the patients' B cells. The expression of this molecule was inducible on the CVID lymphocytes with retinoic acid. Increased spontaneous apoptosis in vitro was observed with the patients' B (23%) and T cells (10%) compared with normal cells (13% and 3%, respectively). Stimulation in vitro with IL-4 and CD40 rescued the B cells from apoptosis and allowed for their differentiation. However, IL-4 plus alpha CD40-driven immunoglobulin production was not quantitatively or qualitatively normal. Failure to overcome apoptosis, a normal step in germinal centre B cell development, may be involved in the lack of differentiation seen in this subset of CVID patients.

ADP-ribosyl Cyclase↗
PubMed1995

Interleukin 4 activates a signal transducer and activator of transcription (Stat) protein which interacts with an interferon-gamma activation site-like sequence upstream of the I epsilon exon in a human B cell line. Evidence for the involvement of Janus kinase 3 and interleukin-4 Stat.

Germ line C transcripts can be induced by IL-4 in the human B cell line, BL-2. Utilizing a IFN-gamma activation site-like DNA sequence element located upstream of the I epsilon exon, we demonstrated by gel mobility shift assays that IL-4 induced a binding activity in the cytosol and nucleus of BL-2 cells. This factor was designated IL-4 NAF (IL-4-induced nuclear-activating factors) and was identified as a tyrosine phosphoprotein, which translocates from the cytosol to the nucleus upon IL-4 treatment. Because these are the characteristics of a signal transducer and activator of transcription (Stat) protein, we determined whether antibodies to Stat proteins will interfere with gel mobility shift and found that antibodies to IL-4 Stat, also known as Stat6, but not antibodies to other Stat proteins, interfere with the formation of the IL-4 NAF complex. Congruous with the involvement of a Stat protein, IL-4 induced robust Janus kinase 3 (JAK3) activity in BL-2 cells. Cotransfection of JAK3 with IL-4 Stat into COS-7 cells produced an intracellular activity which bound the same IFN-gamma activation site-like sequence and comigrated with IL-4 NAF in electrophoretic mobility shift assay. These results show that IL-4 NAF is IL-4 Stat, which is activated by JAK3 in response to IL-4 receptor engagement.

Animals↗
PubMed1995

Germ-line human epsilon heavy chain gene RNA transcripts utilize the full range of alternative 3' splicing seen in productive epsilon mRNA.

An early step in immunoglobulin isotype switching involves the initiation of active transcription of downstream heavy chain loci while they are still in germ-line configuration. This results in the production of 'sterile' germ-line RNA transcripts that do not contain VDJ sequences, the role of which in isotype switching is under intense scrutiny. We investigated whether such human epsilon germ-line transcripts employ the full complement of complex alternative 3' RNA splicing, splicing we have recently reported occurring with productive epsilon mRNA transcripts. Using a reverse transcriptase polymerase chain reaction (RT-PCR) strategy, in which the 5' primer was located in the I region of the epsilon gene, a region expressed in germ-line but not productive (VDJ containing) epsilon transcripts, we showed that the full range of alternative 3' epsilon splices occur with germ-line transcripts. These results demonstrate that epsilon 3' splicing events are independent of 5' isotype DNA switching. Additionally, we showed that, just as with mature epsilon mRNA, the relative production of the various epsilon germ-line mRNA isoforms was responsive to modulation by stimuli such as interleukin-10 (IL-10). Thus B cells, when stimulated to produce epsilon germ-line transcripts, generate a family of germ-line mRNA that differ not only in their initiation sites but, more importantly, also differ in their 3' sequences, sequences that could be important in regulation of the parent gene itself. Furthermore, by discontinuous or trans-splicing, cells could utilize these various epsilon germ-line transcripts to produce the full range of mature IgE proteins prior to undergoing deletional recombination of isotype switching.

Base Sequence↗
PubMed1994

Anti-SCID mouse reactivity shapes the human CD4+ T cell repertoire in hu-PBL-SCID chimeras.

Injecting human peripheral blood mononuclear cells into severe combined immunodeficient (SCID) mice results in long-term engraftment of human lymphocytes, of which > 98% are phenotypically mature, activated T cells. Here we have characterized the human T cells that populate such hu-PBL-SCID chimeras. We report that these human T cells do not mobilize Ca2+ after CD3 stimulation, i.e., their T cell receptor (TCR)-mediated signal transduction is deficient. Chimera-derived human T cells do not secrete lymphokines or undergo blastogenesis after CD3 stimulation, but proliferate in response to interleukin 2 (IL-2), defining the chimera derived human T cells as anergic. Anergy was seen in both the CD4+ and the CD8+ subpopulations. We established human T cell lines from chimeras. These T cells retained their anergic state for 1-2 mo in culture, after which they simultaneously regained the ability to mobilize Ca2+, secrete lymphokines, and to undergo blastogenesis following stimulation via the TCR. Once regaining proliferative responsiveness to CD3 stimulation, these CD4+ T cell lines displayed anti-SCID mouse reactivity and showed no specificity for recall antigens. All CD3-responsive CD4+ T cell clones obtained from such lines were SCID mouse specific, recognizing native major histocompatibility complex class II products on the murine cells. In contrast, chimera-derived human CD8+ cell lines and clones did not display detectable anti-mouse reactivity. The data show that the human T cell system in long term hu-PBL-SCID chimeras is nonfunctional due to both anergy and the limitation of the CD4+ repertoire to xenoreactive clones. The data suggest that long-term hu-PBL-SCID chimerism represents an atypical graft-versus-host reaction in which the human effector T cells become anergic in the murine environment.

Adult↗
PubMed1994

CD58 (LFA-3) stimulation provides a signal for human isotype switching and IgE production distinct from CD40.

Induction of an IgE response involves several discrete steps: 1) induction of epsilon germ line transcription, 2) DNA recombination, and 3) mature RNA transcription/translation. Here we show that ligation of B cell CD58 by CD2, its natural ligand on T cells, or by mAb, provides a novel IL-4-dependent signal for the latter two steps. Highly purified human B cells were induced to produce IgE by costimulation with IL-4 and CD58 mAb. Although CD58 ligation alone was unable to induce epsilon germ-line transcription, in concert with IL-4-stimulated epsilon germ-line transcription it induced the appearance of productive epsilon transcripts and IgE production. The direct involvement of CD2 was demonstrated: B cells cultured with IL-4 plus murine T hybridoma cells transfected with human CD2 produced IgE. A CD40 Fc fusion protein had no effect on CD58-driven IgE production while inhibiting CD40-dependent responses. Furthermore, cells from patients with common variable immunodeficiency produced IgE in response to IL-4 plus CD40 mAb but not to IL-4 plus CD58 mAb. CD58-driven IgE synthesis was IFN-gamma independent and was not enhanced by exogenous IL-6. Functional differences between CD40 and CD58 IgE stimulation were demonstrated. Thus, the CD2:CD58 ligand/counterligand system provides an alternative pathway by which cell contact signaling may regulate IgE. Given the relative importance of CD2 triggering on mucosal T cells and the mucosal location of IgE production, this may be especially true on mucosal surfaces.

Adult↗
PubMed1994

Switch circles from IL-4-directed epsilon class switching from human B lymphocytes. Evidence for direct, sequential, and multiple step sequential switch from mu to epsilon Ig heavy chain gene.

Ig isotype switch via deletional recombination is accompanied by excision of the intervening DNA between the two switch regions. The excised DNA is looped out as extrachromosomal circular DNA or switch circle. Such switch circles have been isolated and characterized in mice. We investigated deletional recombination in human B cells undergoing Ig isotype switching to demonstrate whether switch circles are also excised, and to thereby gain insight into the processes involved in human isotype switching. We characterized the deleted switch circular DNA from IL-4 directed mu to epsilon switching in polyclonal human B lymphocytes. By using two sets of specially designed PCR primers, we amplified switch circle fragments representing switch circles resulting from mu to epsilon direct switching and mu-gamma-epsilon sequential switching. The PCR-amplified products were subcloned by a TA cloning strategy and resulting clones were screened by hybridization with a 5'S epsilon probe. Sequence analysis of the positive clones revealed that all clones representing mu to epsilon direct switching indeed had 5'S epsilon directly joined to 3' S mu. Most clones representing mu-gamma-epsilon sequential switching showed 5'S epsilon joined to 3' S gamma as expected. However, two clones contained S mu and S alpha 1 sequences interposed between 5'S epsilon and 3'S gamma, respectively. These data demonstrate that switch circles are excised during human B cell isotype switching, and that IL-4 directed epsilon class switching is accomplished by 1) direct mu to epsilon switching, 2) sequential mu-gamma-epsilon switching, and 3) double sequential mu-alpha-gamma-epsilon switching.

B-Lymphocytes↗
PubMed1994

The SCID mouse environment causes immunophenotypic changes in human immature T-cell lines.

In order to evaluate whether the SCID mouse can provide the microenvironment for the growth of human immature T-cell leukemias and if in vivo growth alters their phenotype, we examined the behavior of 3 well-characterized T-cell acute lymphoblastic leukemia (T-ALL)-derived T-cell lines which are at different stages of maturation (CEM, SUP-T3 and MOLT-4f) after transfer to non-irradiated SCID mice. All 3 T-cell lines engrafted and proliferated to form tumors in the mice and showed dissemination patterns in the SCID mouse comparable to those of T-ALL in man: i.e., human cells were detectable by flow cytometry or were cultured from mouse bone marrow, spleen, liver or thymus. CEM, which is the most immature T-cell line, readily formed tumors after injection of cells. The more mature T-cell lines, SUP-T3 and MOLT-4f, required a longer time period, even after injection of higher cell numbers. Whereas no changes in the configuration of the rearranged T-cell receptor genes were detected, striking phenotypic changes were observed in all 3 leukemias growing in the SCID mice after injection. SCID-CEM cells showed an increase in the surface expression of CD3 and CD8 and a decrease in the expression of CD1 and CD71 (transferrin-receptor). SCID-MOLT-4f cells showed an increase in CD5 and CD8 expression and a decrease in CD45RA expression. SCID-SUP-T3 cells showed increased expression of CD8 and CD45RA. Apparently, the mouse environment caused changes in cell-surface antigen expression on the T-ALL-derived T-cell lines. Some immunophenotypic changes remained stable during subsequent growth in culture of SUP-T3 cells, suggesting that maturation of the cell line occurred in vivo. The other cell lines, CEM and MOLT-4f after undergoing in vivo-induced changes, reverted to the original immunophenotype, suggesting transitory activation in vivo. These data point out the importance of stromal factors in defining growth and maturation of human leukemic cells in vivo, in SCID mice.

Animals↗
PubMed1994

Complex alternative RNA splicing of epsilon-immunoglobulin transcripts produces mRNAs encoding four potential secreted protein isoforms.

In RNA from human IgE-producing lymphocytes, we previously discovered two alternatively spliced epsilon-immunoglobulin mRNA isoforms that encode a novel secreted form of IgE and a membrane-bound species. Further analysis using epsilon-specific reverse transcriptase polymerase chain reactions has elucidated several additional alternatively spliced species of epsilon mRNA. One RNA isoform is generated by splicing the CH4 exon to a novel distal splice acceptor site, forming an epsilon RNA species (designated CH4-M2") that encodes a secreted epsilon protein 6 amino acids larger than the classic secreted epsilon protein. The other three novel epsilon RNAs are generated by splicing from within CH4 to a new exon structure (designated CH5) that is located between CH4 and the membrane exons. Since the three new mRNAs using CH5 share the same stop codon in CH5, they all encode the same novel protein, which is 10 amino acids shorter than the classic secreted epsilon heavy chain. The new alternatively spliced epsilon mRNAs reported here, in addition to the previously reported forms encoding membrane and larger secreted IgE, appear to reflect the normal splicing pattern in humans, as we have detected all these epsilon RNAs in all the human IgE-secreting cells and cell lines tested.

Alternative Splicing↗
PubMed1994

Acute bone marrow toxicity and pancytopenia following exposure to lead chromate, xylene, and ethylbenzene in a degloving injury.

A case of unique acute bone marrow toxicity and pancytopenia following subcutaneous exposure to lead chromate, xylene, and ethylbenzene in a previously healthy patient is reported. The patient sustained an extensive degloving injury to his lower extremity. The wound was contaminated with traffic paint containing lead chromate pigment along with a large volume of xylene and ethylbenzene solvent. Consequences of the patient's clinical course and management of degloving injuries are discussed.

Accidents, Traffic↗
PubMed1994

B cell dysfunction after bone marrow transplantation is associated with decreased Ca2+ flux upon membrane Ig crosslinking.

Patients undergoing bone marrow transplantation have a long-lasting defect of B cell-mediated immunity. Both quantitative (decreased blood B cell counts) and qualitative (decreased Ig production) abnormalities of B cells have been described. To better understand the mechanism of the qualitative defect and its potential relation to B cell immaturity, we studied the in vitro responsiveness of B cells to polyclonal stimuli in patients at 2-12 months post-transplant and in normal neonates. Several key steps of the B cell program were deficient in the patients while they were relatively normal in the neonates. These included (i) early activation as assessed by Ca2+ flux; (ii) late activation as assessed by the increase in cell size and upregulation of the activation antigens CD25 and CD71; and (iii) proliferation as assessed by the number of cycling cells after stimulation. We conclude that the functional B cell defect during the early (< 1 year) post-transplant period extends back to the level of early activation and cannot be simply attributed to the relative immaturity of post-transplant B cells.

Adolescent↗
PubMed1994

Prevalence and epidemiologic correlates of human T cell lymphotropic virus infection among intravenous drug users.

Intravenous drug users (IVDUs) in Seattle (n = 213) were studied to identify the prevalence and predominant types of and risk factors for human T cell lymphotropic virus (HTLV) infection. Detailed questionnaires, serologic screening, and polymerase chain reaction analysis (for a subset) were used. Evidence of HTLV infection was found in 16.5%, of which 89% were HTLV-II. HTLV infection was significantly associated with nonwhite race, older age, more years of intravenous drug use, prior use of heroin, history of gonorrhea, history of any sexually transmitted disease, hepatitis B virus infection, and antibody to herpes simplex virus type 2 (HSV-2). By stepwise logistic regression analysis, associations persisted with race, age, hepatitis B markers, and HSV-2. Thus, the strong association of HTLV with hepatitis B, a marker for injection behavior, and the independent association with HSV-2 infection, a sexually transmitted pathogen, suggest similarities in the epidemiology of HTLV and human immunodeficiency virus infections in IVDUs.

Adult↗
PubMed1994

Diesel exhaust particles induce local IgE production in vivo and alter the pattern of IgE messenger RNA isoforms.

Diesel exhaust particles (DEP) have been implicated in the increased incidence of allergic airway disorders. We investigated the effects of DEP on localized immunoglobulin production by performing nasal challenges with varying doses of DEP and analyzing the local immune response in nasal lavages obtained before and after. A significant rise in nasal IgE but not IgG, IgA, IgM, or albumin was observed in subjects 4 d after challenge with 0.30 mg DEP, equivalent to exposure on an average Los Angeles day. Direct evidence for DEP-enhanced local production of IgE was that challenge increased the number of IgE-secreting cells in lavage fluid from < 1 in 2,000,000 to > 1 in 100,000 but did not alter the number of IgA-secreting cells. There was a concomitant increase in epsilon mRNA production in the lavage cells. Additionally, DEP altered the relative amounts of five different epsilon mRNAs generated by alternative splicing, mRNAs that code for different IgE proteins. These results show that DEP exposure in vivo causes both quantitative and qualitative changes in local IgE production. The implication is that natural exposure to DEP may result in increased expression of respiratory allergic disease.

Adult↗
PubMed1994

Abundance of a restricted fetal B cell repertoire in marrow transplant recipients.

Patients undergoing bone marrow transplantation are humorally immunodeficient for one or more years post-transplant. This immunodeficiency could be partially caused by B cell repertoire restriction similar to that observed in ontogeny. To test this idea, the abundance of rearranged genomic segments bearing five variable heavy chain (VH) genes was compared in patients at several timepoints post-transplant and in immunologically normal neonates, infants and adults. The genes evaluated in the study (VH6, VH4-58p2, VH3-56p1, VH3-20p1 and VH3-13-2) were selected from those commonly utilized by fetal B cells. The assay employed quantitative PCR and oligonucleotide hybridization detection under conditions designed to detect relatively unmutated forms of these genes. In blood B cells from early post-transplant (2-5 months) patients, these VH genes were markedly overutilized compared with normal adults. B cells from late post-transplant (6-21 months) patients and from normal neonates and infants also overutilized these genes; however, to a lesser degree than early post-transplant B cells. The data suggest that, as in ontogeny, the B cell repertoire is strikingly restricted to fetal-type VH genes early post-transplant, and may become normal only very late (years) post-transplant.

Adult↗
PubMed1993

Aspartame is no more likely than placebo to cause urticaria/angioedema: results of a multicenter, randomized, double-blind, placebo-controlled, crossover study.

BACKGROUND: Anecdotes and single case reports have suggested that the high-intensity sweetener, aspartame, may be associated with allergic/hypersensitivity-type reactions. METHODS: We conducted a multicenter, placebo-controlled clinical study to evaluate individuals who had experienced urticaria and/or angioedema allegedly associated with ingestion of an aspartame-containing product. Despite extensive recruiting efforts over 4 years, only 21 subjects could be enrolled. After admission to clinical research units, subjects were given aspartame and placebo in a randomized, double-blind, crossover fashion. Subjects received, on different days, increasing doses (50, 300, 600 mg) of aspartame and placebo at 8:00 AM, 10:00 AM, and noon. Subjects who weighed less than 40 kg received one half of these doses. Conversion products of aspartame, aspartyl-phenylalanine diketopiperazine and beta-aspartame, were also included in the aspartame arm of the study. Positive reactions were defined as urticaria (hives with wheals 4 mm or more in diameter with a collective diameter of at least 15 mm or one or more hives with a wheal of 4 mm or greater with a flare of 8 mm or greater) or as angioedema. RESULTS: According to these criteria, four reactions were observed; two followed aspartame ingestion and two followed placebo ingestion (p = 1.00). The incidence of other adverse experiences was no different after aspartame versus placebo ingestion (p = 0.289). CONCLUSION: These results indicate that aspartame and its conversion products are no more likely than placebo to cause urticaria and/or angioedema reactions in subjects with a history consistent with hypersensitivity to aspartame.

Administration, Oral↗