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Biomedical subjects

J M Green

Publications and source records attributed to J M Green.

At least 73 records · Page 4Linked to original sources
PubMed1996

Diagnosis of Duchenne muscular dystrophy: parents' experiences and satisfaction.

The purpose of the study was to investigate parents' experiences of diagnosis of Duchenne muscular dystrophy. Data were collected retrospectively by postal questionnaire from the parents of 158 boys diagnosed at different times and locations and, for some, after lengthy periods of uncertainty. Most parents knew nothing about Duchenne before being given the diagnosis. There was considerable variation in parents' experiences but the only independent predictors of parents' satisfaction with how the diagnosis was given were obtaining the information that they wanted and feeling that they had understood and remembered it. What distinguishes Duchenne from, say, Down syndrome is that doctors are having to deliver a death sentence on a child. Our data show that this can be done in a way that is satisfactory to parents. What is required is empathy and sensitivity to parents' informational and emotional needs.

Adult↗
PubMed1995

B7/CD28-dependent and -independent induction of CD40 ligand expression.

The induction of a T cell-dependent Ab response is mediated by the interaction of the T cell activation Ag, CD40 ligand (CD40L), with CD40. Since this interaction is independent of Ag, coreceptors such as CD4, and MHC molecules, the expression of the CD40L must be strictly regulated or B cell-mediated autoimmunity may be produced. In this study, we examined the requirements for costimulatory signals for induction of CD40L expression in vitro and in vivo on CD4+ T cells from normal and CD28-deficient mice following stimulation with anti-CD3, Con A, or specific peptide Ag. Expression of B7-1 was both necessary and sufficient for induction of the CD40L on normal CD4+ T cells when L cell transfectants were used as APCs. When normal accessory cell populations were used, only partial inhibition of induction of the CD40L was observed with reagents that inhibit B7/CD28 interactions. Furthermore, the CD40L could be induced on CD4+ T cells from CD28-deficient mice. Thus, non-B7/CD28 cellular interactions can also mediate the costimulatory signals needed for induction of CD40L expression.

Animals↗
PubMed1995

The intracellular transport of MHC class II molecules in the absence of HLA-DM.

The HLA-DM alpha and HLA-DM beta genes encode a nonpolymorphic, class II-like molecule that functions by an as yet undefined mechanism in the assembly of processed antigen-HLA class II complexes. Mutant cells that fail to express HLA-DM are deficient in Ag processing. We previously isolated a subcellular compartment in mouse B cells in which functional processed Ag-class II complexes are first formed, referred to as the peptide-loading compartment. Here, evidence is provided that HLA-DM resides in a subcellular compartment with the characteristics of a peptide-loading compartment in a human B lymphoblastoid cell line, but is not required for the intracellular transport of HLA-DR3 molecules to a corresponding compartment in HLA-DM-deficient cells. Thus, the primary defect in HLA-DM-deficient cells does not appear to be a failure in the intracellular trafficking of class II molecules.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1995

CD43 is a murine T cell costimulatory receptor that functions independently of CD28.

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.

Animals↗
PubMed1995

HER4 expression correlates with cytotoxicity directed by a heregulin-toxin fusion protein.

We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2. All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected. HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.

ADP Ribose Transferases↗
PubMed1995

Paratope characterization by structural modelling of two anti-cortisol single-chain variable fragments produced in E. coli.

Two monoclonal antibodies (mAbs), 5A4 and 6D6, directed against cortisol, have been obtained; 6D6 is used in an assay kit for cortisol. The antibodies also recognize other, structurally related steroids present in the sample assayed. To improve the specificity of the assay, we aimed to minimize the recognition of non-cortisol steroids by the two anti-cortisol mAbs. Our strategy consisted in constructing an efficient expression vector in E. coli which produced the single-chain variable fragment (scFv) of the mAbs in the periplasmic space. We demonstrated that temperature and inducer concentration of the bacterial culture influenced dramatically the yield of active scFv. From the nucleotide sequence we constructed a three-dimensional model of the two variable fragments in order to understand why related steroids are, or are not recognized by the antibody. For both antibodies, we have identified chemical groups which are probably involved in the binding of the steroid haptens and the antibodies. The hydrophobic pocket formed by the antibody comprises two or three tryptophan residues which can interact with the steroid nucleus by stacking. The serine at position 35 of the heavy chain is buried in the back of the pocket and can form a hydrogen bond with the 20-keto group of the cortisol. The stacking interactions and the hydrogen bond orient the steroid in the pocket. This reactivity of the binding site is sustained by the analysis of the cross-reactions of related steroids with the mAbs.

Amino Acid Sequence↗
PubMed1995

Generation and characterization of monoclonal antibodies specific for members of the mammalian 70-kDa heat shock protein family.

The 70-kDa heat shock proteins (hsp70) are a highly conserved, abundant, and ubiquitous family of proteins expressed by all organisms from bacteria to humans. It is well established that hsp70 family members function as molecular chaperones and aid in the intracellular folding of newly synthesized or denatured proteins. Current evidence suggests an emerging role for hsp70 family members in immune responses and in clinically important responses to stress and tissue damage. Here we report the generation and characterization of several MAbs to hsp70 family members. Immune responses to this highly conserved family were induced in mice by immunization with synthetic peptides that contain regions of the mouse mitochondrial hsp70 coupled to a potent helper T cell epitope derived from tetanus toxoid. The resulting MAbs include ones specific for the human and mouse mitochondrial hsp70 and others that show cross-reactivity among the family members and recognize the mitochondrial hsp70, the endoplasmic reticulum resident hsp70, Bip/grp78, the constitutively expressed cytosolic hsp70, hsc70, and the heat-induced member, hsp70. Significantly, these MAbs are effective in Western blotting, in immunoprecipitation, and in immunofluorescence, and thus should find applications in the purification and detection of members of this important family.

Amino Acid Sequence↗
PubMed1995

Obstetricians' views on prenatal diagnosis and termination of pregnancy: 1980 compared with 1993.

OBJECTIVE: To examine the attitudes of obstetricians in England and Wales concerning prenatal diagnosis and termination of pregnancy and to compare these with a similar survey carried out in 1980. METHODS: Postal questionnaires, based on those used in 1980, were sent to a random sample of 555 nonacademic consultant obstetricians, 71% of whom replied. RESULTS: The proportion of consultant obstetricians with a conscientious objection to termination of pregnancy was the same in both surveys, although virtually all conscientious objectors said that they made an exception for serious fetal abnormality. A major change was in the proportion saying that they generally required an undertaking to terminate an affected pregnancy before proceeding with amniocentesis: 75% in 1980 compared with 34% in 1993. As in 1980, lower priority is given to telling women of negative test results than of positive. More obstetricians in 1993 are prepared to consider terminations later in pregnancy, both for fetal abnormality and for social reasons, than was the case in 1980, but many are not prepared to recommend termination as late in pregnancy as the law allows. In some cases this is due to misapprehensions about what is legal. There are particular problems concerning terminations for fetal anomalies that not all would consider serious. One in seven of the 1993 sample would not recommend termination for cystic fibrosis at any gestation. Only 13% would recommend termination for Down's syndrome beyond 24 weeks. There is a need not only for clarification of the law, but also of obstetrician's willingness to implement it.

Abortion, Induced↗
PubMed1995

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.

4-Aminobenzoic Acid↗
PubMed1995

Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis.

Abnormal growth of airway smooth muscle may play an important role in the pathogenesis of human airway diseases. Little is known about the proliferative responses of cultured airway smooth muscle cells, nor of the precise pathways responsible for mitogenesis in these cells. We assessed DNA synthesis, cell proliferation, and mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes after exposure to four potential mitogens: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and 5-hydroxytryptamine (5-HT). Stimulation with either PDGF or IGF-1 induced substantial increases in DNA synthesis and cell number, as reflected by [3H]thymidine incorporation, flow cytometry, and methylene blue staining. Treatment with EGF or 5-HT, on the other hand, induced only modest DNA synthesis and no increase in cell number. Immunoblots and kinase renaturation assays of cell extracts demonstrated activation of both the 42- and 44-kDa MAP kinases within minutes of either PDGF, IGF-1, EGF, or 5-HT exposure. However, relative to EGF and 5-HT stimulation, late-phase MAP kinase activation was significantly greater after treatment with the mitogens PDGF and IGF-1. We conclude that in cultured bovine tracheal myocytes 1) PDGF and IGF-1 are potent mitogens; 2) MAP kinase may be activated subsequent to stimulation of either receptor tyrosine kinases (PDGF, EGF, IGF-1) or G protein-linked receptors lacking in known tyrosine kinase activity (5-HT); and 3) unsustained MAP kinase activation is insufficient for mitogenesis. Finally, the finding that mitogenicity correlates with the late phase of MAP kinase activation is consistent with the notion that sustained MAP kinase activation is important for bovine tracheal myocyte proliferation.

Animals↗
PubMed1995

Migration and proliferation of guinea pig and human airway epithelial cells in response to tachykinins.

Restoration of the epithelial lining of a damaged airway is a necessary component of airway repair. Tachykinins, including substance P (SP) and neurokinin A (NKA), are localized to sensory nerves within the airway mucosa. These tachykinins regulate several airway functions, but their role in the repair of the epithelium has not been explored. To determine whether tachykinins stimulate migration and proliferation of airway epithelial cells, guinea pig tracheal epithelial (GPTE) and human bronchial epithelial (HBE) cells were grown in primary culture for 4-5 days. Epithelial cell migration was assessed in a blindwell chemotaxis chamber, and proliferation was determined by immunohistochemistry after incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). Both GPTE and HBE cells migrated after stimulation with 10(-11) M NKA [23.0 +/- 3.6 vs. 5.4 +/- 1.2 cells per 10 high-power fields (hpf), P < 0.001, n = 8 for GPTE cells; 18.4 +/- 2.3 vs. 3.8 +/- 0.5 cells per 10 hpf for control, P < 0.001, n = 4 for HBE cells]. Migration was stimulated within 2 h, was maximal after 6 h, and was attenuated substantially by the neurokinin 2 (NKA)-receptor antagonist SR-48968. NKA-stimulated migration was both chemokinetic and chemotactic, and it could be blocked by inhibition of protein synthesis with cyclohexamide, inhibition of microtubular function with colchicine, or inhibition of actin microfilament elongation with cytochalasin D.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Nonreamed interlocked intramedullary tibial nailing. One community's experience.

Forty-nine acute displaced tibial fractures (31 closed, 18 open: 5 Grade I, 7 Grade II, 4 Grade IIIA, and 2 Grade IIIB) were treated in 1 community with a standard operative protocol using a distractor without a fracture table, and an unreamed interlocked tibial nail. Forty-six fractures healed (94%). Complications included 3 nonunions (6%), 2 deep infections (4%), 9 delayed unions (18%), 4 angular malunions (8%), 2 rotatory malunions (4%), and 12 interlocking screws bent or broke (24%). Twenty-eight patients (57%) required at least 1 additional operation to obtain union, most commonly dynamization of a statically locked nail. The authors conclude that unreamed tibial nails provide adequate stabilization of displaced tibial fractures and can be used in the management of most open or closed tibial fractures. However, static locking is required in axially unstable fractures. Early dynamization or exchange nailing and bone grafting should be considered to hasten union and avoid screw failure. The distractor is an excellent adjunctive technique for reduction and alignment of tibial shaft fractures during intramedullary nailing.

Adolescent↗
PubMed1995

The intracellular assembly of antigenic-peptide-class II complexes.

The immune system employs remarkable strategies to ensure that foreign antigens, from the most complex pathogens to the simplest proteins, are displayed on the surfaces of cells which are targets of T lymphocyte recognition. At the heart of these strategies is the molecular transformation of a soluble protein antigen to a complex of a small peptide containing the antigenic determinant bound to a cell surface Major Histocompatibility Complex class I or class II protein. This process is termed antigen presentation. Progress in a variety of laboratories over the last several years has yielded a wealth of information about the molecular mechanisms underlying antigen presentation, providing potential new approaches to vaccine design. Here we describe recent studies in our laboratory aimed at elucidating the intracellular site in B lymphocytes in which antigenic peptide-class II complexes are assembled for recognition by helper T cells and the regulation of this assembly process. Our results suggest that processed antigen-class II complexes are assembled in a unique compartment in the endocytic route which contains all the necessary cellular and molecular machinery for assembly and that B cells regulate the assembly process in response to external and internal signals.

Antigen-Presenting Cells↗
PubMed1994

Serum screening for Down's syndrome: experiences of obstetricians in England and Wales.

OBJECTIVES: To assess the experiences of obstetricians in England and Wales of serum screening for Down's syndrome. DESIGN: Postal questionnaire survey. SUBJECTS: Questionnaires were sent to all practising obstetricians in England and Wales with nonacademic appointments who had not participated in an earlier (randomly sampled) survey of obstetricians' attitudes (n = 555). Responses were received from 393 (71%), of which 351 were analysed. The data represent about one third of obstetric consultants in England and Wales. MAIN OUTCOME MEASURES: The extent of use of serum screening for Down's syndrome, and the problems encountered. RESULTS: Serum screening for Down's syndrome was being offered on some basis by virtually all obstetricians in the survey. Nearly half the sample said that they did not have adequate resources for counselling all the women to whom screening was offered. Many problems were reported, which in all cases were more common than equivalent problems encountered with serum screening for neural tube defects. Over 80% (289) said that women not understanding the test was a problem. CONCLUSIONS: There is considerable confusion associated with serum screening for Down's syndrome. The precedent of serum screening for neural tube defects does not seem to have lessened the problems experienced, rather the contrary. Many obstetricians report inadequate resources for counselling, which is consistent with the high prevalence of problems associated with women not understanding the test. There is an urgent need to consider what counselling should consist of and who should undertake it and to ensure that necessary resources are available.

Anxiety↗
PubMed1994

Keratinocyte-derived T cell costimulation induces preferential production of IL-2 and IL-4 but not IFN-gamma.

By using superantigens, we have found previously that keratinocytes activated by IFN-gamma could serve as accessory cells, providing costimulatory signals needed to induce T cell proliferation. Here, we compared the profile of cytokines produced by T cells stimulated in the presence of activated keratinocytes with the response seen using professional APCs. When keratinocytes are used as accessory cells there is a specific defect in T cell IFN-gamma production, whereas IL-2 and IL-4 are induced at levels comparable with those seen when professional APCs are used as accessory cells. Because keratinocytes express BB-1, a CD28-ligand distinct from B7-1 or B7-2 (which are found on professional APCs), we examined the possibility that the defect in IFN-gamma production might be a result of nonproductive CD28 engagement. However, even when the CD28 pathway is directly activated by a stimulatory mAb, there is no induction of IFN-gamma production in keratinocyte-supported cultures. In these same cultures IL-2 production is increased 10-fold, thus demonstrating a specific deficiency in the induction of IFN-gamma rather than a failure to respond to CD28 stimulation. Analysis by reverse transcriptase-PCR and ELISA for the inducible p40 chain of IL-12 reveals that keratinocytes produce little if any messenger RNA and no protein for IL-12 p40 compared with professional APCs. Addition of rIL-12 to keratinocyte-supported cultures restores IFN-gamma levels to those seen when professional APCs are present. Finally, when T cells are restimulated and analyzed at later time points (10 to 14 days) we find a refinement in cytokine profiles: T cells stimulated in the presence of professional APCs produced the Th1 cytokines IL-2 and IFN-gamma, whereas T cells stimulated in the presence of activated keratinocytes produced only the Th2 cytokine IL-4. The specific ability of keratinocytes to induce a Th2 response seems most closely linked to their absence of IL-12 production, and may be important in the maintenance of peripheral tolerance to self-Ags or in the immune response to exogenous Ags, pathogens, or haptens encountered in skin.

Antigen-Presenting Cells↗
PubMed1994

T cell receptor stimulation, but not CD28 costimulation, is dependent on LFA-1-mediated events.

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. In this manuscript we specifically examine the role of two accessory molecules, CD28 and LFA-1, in modulating the T cell proliferative response to a variety of stimuli. We demonstrate that the proliferation induced by staphylococcal enterotoxins A and B in combination with CD28 costimulation is dependent on LFA-1-mediated events. This requirement for LFA-1 is independent of T cell-accessory cell adhesion. Similarly, an allogeneic mixed lymphocyte reaction, which has previously been shown to be a CD28-dependent response, can be inhibited by blockade of LFA-1. This suggests LFA-1 plays an essential role in these responses, either by enhancing intercellular adhesion or by an independent signal transduction event. In contrast, when the primary activating stimulus is delivered by immobilized anti-CD3 antibody or by PMA, and the secondary stimulus by either alpha-CD28 or cell-bound CD28 ligand B7, there is no requirement for LFA-1. In addition, we demonstrate that cross-linking of LFA-1 with immobilized monoclonal antibody, or engagement of LFA-1 with ICAM-1 expressed on the surface of a CHO cell, provide an insufficient costimulus for T cell proliferation initiated by enterotoxin, immobilized alpha-CD3 or phorbol ester. Our data suggests that LFA-1, in contrast to CD28, functions not as a costimulatory molecule, but serves primarily to modulate the signal delivered through the T cell receptor.

Animals↗
PubMed1994

Modulation of T cell proliferative response by accessory cell interactions.

Antigen-specific activation of the T cell is accomplished by engagement of the T cell receptor (TCR) by an antigen (Ag)/MHC complex presented on the surface of an antigen- presenting cell (APC). However, it has been demonstrated that engagement of the TCR by Ag/HC complexes alone is normally insufficient to lead to a proliferative response and the development of effector function. Thus it has been proposed that the APC also provides additional signals which serve to modulate the T cell's response. These second or costimulatory signals are thought to be critical in the generation of a T cell-driven immune response. Several receptors have been proposed to be capable of serving as costimulatory receptors. Candidate molecules include CD28 and LFA-1 as well as other receptors. In this review the studies that we have performed to clarify the role of both LFA-1 and CD28 in providing costimulatory activity for T cell activation are discussed. In addition, we present evidence that under certain conditions, TCR signalling alone can be sufficient to lead to T cell proliferation.

Animals↗