An improved method for the detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization.
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Amniotic Fluid↗
Biomedical subjects
L Barber
Publications and source records attributed to L Barber.
Systemic hypertension and hypercholesterolemia in vein grafts: effects on the function and morphology of experimental vein grafts.
Hypertension and hypercholesterolemia are known risk factors for the development of atherosclerosis and are considered to influence the development of vein graft intimal hyperplasia. This study examines the effect of hypertension (two-kidney, one-clip model for 8 weeks) and hypercholesterolemia (1% cholesterol diet for 8 weeks) individually and in combination on the formation of intimal hyperplasia and the vasomotor function of vein grafts. Forty New Zealand White rabbits underwent a carotid vein bypass graft. Ten were controls, 10 were hypertensive, 10 were hypercholesterolemic, and 10 had both hypertension and hypercholesterolemia. Hypertension and/or hypercholesterolemia were present for 4 weeks prior to and for 4 weeks after surgery. All vein grafts were harvested at 4 weeks postoperatively for histology (n = 6) or contractility studies (n = 4). Compared to controls, hypercholesterolemia increased intimal but not medial thicknesses of the vein grafts and enhanced smooth muscle cell contractility. Hypertension did not increase vein graft intimal or medial thicknesses but did augment vein graft contractility compared to controls. Hypertension with hypercholesterolemia increased intimal and medial thicknesses and enhanced vasoreactivity in vein grafts. The results show that hypertension influences the vasoreactivity but not the development of intimal hyperplasia in vein grafts. When hypertension is combined with hypercholesterolemia, there is both an increase in the medial thickness and an attenuation of vasomotor function compared to hypercholesterolemia alone, although there is no further increase in intimal hyperplasia formation. Therefore, this study suggests that the combination of both atherogenic risk factors does not act synergistically in promoting either the formation of intimal hyperplasia or vasomotor dysfunction in vein grafts.
Animals↗
Diabetes mellitus and experimental vein graft structure and function.
PURPOSE: Diabetes mellitus is a known risk factor for accelerated atherosclerosis and for postangioplasty restenosis. METHODS: This study examines the effect of chronic, uncontrolled, alloxan-induced diabetes on the structure and vasomotor function of vein bypass grafts in 20 male New Zealand white rabbits with diabetes and in 10 controls. After 8 weeks of diabetes, a common carotid vein bypass graft was performed. Four weeks after operation, vein grafts and contralateral jugular veins were harvested. RESULTS: Diabetes induced a twofold increase in the vein graft intimal thickness compared with control. There was no change in medial thickness. Electron microscopy of the vein grafts in diabetes revealed intercellular gaps in the endothelium lining and abnormal endothelial cell junctions compared with controls. Diabetes significantly increased the maximal contractions generated in vein grafts to all contractile agonists tested without any change in sensitivity. CONCLUSIONS: This study demonstrates that diabetes alters endothelial cell structure and increases the development of intimal hyperplasia with increased maximal contractility in vein grafts and therefore suggests that the vein grafts in diabetes are more susceptible to early stenosis.
Animals↗
Control of the structural and functional consequences of vein graft intimal hyperplasia with a 21-aminosteroid--U74389G.
Following angioplasty and vein bypass grafting, there is endothelial cell injury, infiltration of leukocytes and smooth muscle cell (SMC) proliferation leading to intimal hyperplasia which may result in stenosis and can lead to eventual occlusion. This study examines the effect of the 21-aminosteroid U74389G (Upjohn Company), on the formation of vein graft intimal hyperplasia in vivo and on SMC DNA synthesis and proliferation in vitro. Twenty New Zealand White rabbits had a right carotid interposition bypass graft using the ipsilateral external jugular vein. Ten animals received chronic oral therapy with U74389G (25 mg/kg/day; begun 5 days before surgery and continued until harvest) and 10 control animals received vehicle only. All animals were sacrificed on the 28th postoperative day. Vein grafts were harvested either for histology/videomorphometry (n = 6 per group) or for in vitro isometric tension studies (n = 4; four 5 mm rings per graft). The incorporation of [3H]thymidine into the cellular DNA of serum-stimulated rabbit aortic SMC (passage 6th to 12th) was assessed in the presence of increasing concentrations of U74389G (10(-9) to 10(-4) M). The effect of U74389G on in vitro cell proliferation was also assessed. Treatment with U74389G produced a 44% decrease in overall mean intimal thickness from 82 +/- 1 microM (mean +/- S.E.M.) in the controls to 57 +/- 10 microM in the U74389G treated vein grafts (p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
FISH detection of trisomy 21 in interphase by the simultaneous use of two differentially labelled cosmid contigs.
Techniques have been reported in which fluorescence in situ hybridisation (FISH) and cosmid probes are used to detect trisomy 21 (and other abnormalities involving chromosomes X, Y, 13, and 18) on uncultured amniocytes. However the detection rate of trisomy 21 is lower than for the other anomalies owing to a larger number of uninformative results and false negatives. We report the simultaneous use of two differentially labelled cosmid contigs to improve the detection rate of trisomy 21 on uncultured amniocyte samples thus allowing the prenatal diagnosis of Down's syndrome even if only few labelled nuclei are available.
Amniotic Fluid↗
Pretreatment with cytosine arabinoside does not protect against the delayed effects of irradiation on thrombopoiesis and erythropoiesis.
We have examined the capacity of the thrombopoietic and erythropoietic systems to respond to challenge with the cytotoxic drug 5-fluorouracil (5-FU) following sublethal doses of irradiation. Normal adult mice respond to 5-FU with a mild thrombocytopenia followed by a marked rebound thrombocytosis. One month after 2 Gy whole-body radiation, platelet counts took longer than normal to reach a nadir after 5-FU, and the rebound thrombocytosis was reduced. A normal response was seen when the interval after radiation was extended to 4 months. Increasing the radiation dose to 6.5 Gy resulted in a much smaller rebound thrombocytosis when 5-FU was given 1 month later. Extending the interval before the drug was given resulted in a normal response being regained between 4 and 7 months. Erythrocyte levels were temporarily depressed after 5-FU in all mice, and it took longer for recovery to occur if they had been irradiated. In another series of experiments, we investigated the effect of priming the mice before irradiation to see if this resulted in radioprotection. An injection of cytosine arabinoside (Ara-C) 2 days before a dose of 6.5 Gy resulted in the expected earlier recovery in platelet counts. To see if cells of the megakaryocyte lineage were protected from the delayed effects of irradiation by this treatment, mice were given 5-FU 1 month after Ara-C plus irradiation. The period of thrombocytopenia was followed by only a small rebound thrombocytosis, and platelet counts were indistinguishable from those found for mice not primed with Ara-C before irradiation. These experiments revealed a delayed effect of irradiation on the thrombopoietic and erythropoietic systems, which was long-lasting but not permanent at the doses used. The effects were not eliminated by priming mice with Ara-C before irradiation.
Animals↗
Postoperative reduction of high serum cholesterol concentrations and experimental vein bypass grafts. Effect on the development of intimal hyperplasia and abnormal vasomotor function.
Hypercholesterolemia is an important contributor to the development of intimal hyperplasia and superimposed accelerated atherosclerosis in vein bypass grafts. This study examines the effect of dietary modification of serum cholesterol on the development of intimal hyperplasia and vasomotor function of vein grafts. Thirty male New Zealand White rabbits had a right carotid vein bypass graft and were put to death 28 days after the operation. Twenty animals received a 1% cholesterol diet for 4 weeks before the operation. In 10 animals this diet was continued until harvest (hypercholesterolemia group). In another 10 animals the diet was changed to standard rabbit chow on the day of the surgical procedure and continued until harvest (cholesterol reduction group). The last 10 animals were control subjects. Vein grafts were harvested either for histologic study or for in vitro isometric tension studies. Cumulative dose response curves to norepinephrine, serotonin, bradykinin, and endothelin-1 were determined. After in situ pressure fixation, intimal thicknesses of the vein grafts were measured by videomorphometry. The change in diet produced a 74% reduction in serum cholesterol concentration within 28 days. There was a 26% reduction in the intimal thickness of vein graft intimal hyperplasia and the macroscopic disappearance of atheromatous lesions from the graft wall, which are always observed in vein grafts from the hypercholesterolemia group. Cholesterol reduction did not change hypercholesterolemia-induced agonist supersensitivity. Therefore, cholesterol reduction slows the formation of intimal hyperplasia in vein grafts but does not prevent the persistence of the hypercholesterolemia-associated smooth muscle phenotype.
Animals↗
Hypercholesterolemia and experimental vein grafts: accelerated development of intimal hyperplasia and an increase in abnormal vasomotor function.
Hypercholesterolemia is an important risk factor for the development of atherosclerosis. Late vein graft failure has been attributed to a combination of both intimal hyperplasia and atherosclerosis. This study examines the effect of hypercholesterolemia on the early morphology and vasomotor function of experimental vein grafts. Forty New Zealand White rabbits received either a 1% cholesterol diet (n = 24; HC) or a standard diet (n = 16; CON) for 4 weeks before operation and thereafter until harvest. All animals underwent a reversed vein common carotid artery bypass. The vein grafts and contralateral veins were harvested at 2 and 4 weeks after operation in both groups for either histological and morphometric analysis (n = 8 for each group) or for in vitro isometric tension studies using serotonin (5-HT), norepinephrine (NE), bradykinin (BK), and endothelin-1 (ET) and following NE precontraction, relaxation in response to acetylcholine (ACh) and sodium nitroprusside (SNP). Serum cholesterol levels were measured after 4, 6, and 8 weeks of the cholesterol diet. Serum cholesterol concentrations were 20 to 30 times higher than controls in the hypercholesterolemic animals at all times. The intimal area of the grafts in the HC group increased by twofold at 2 weeks and threefold at 4 weeks compared to corresponding controls. In contrast to the CON vein grafts, the intima of the vein grafts from HC consisted mainly of lipid-laden smooth muscle cells with scattered interspersed macrophages and occasional lipid plaques between the intima and the media. Medial areas were similar in all grafts. HC grafts became progressively more sensitive to 5-HT at 2 and 4 weeks. A supersensitivity to NE and BK developed at 4 weeks in HC grafts. There was no change in sensitivity to ET. While no graft relaxed to ACh, HC grafts contracted at low doses. All grafts responded to SNP in a dose-dependent manner. In contrast to CON veins, HC veins demonstrated an increase in sensitivity to NE and a contractile response to 5-HT (at 4 weeks only). HC veins did not relax in a dose-dependent manner in response to ACh. No changes in the morphology of HC veins were noted. HC produces changes in the structure and associated vasomotor abnormalities of vein grafts. Closely related functional changes also occur in contralateral veins. HC appears to induce intrinsic changes in smooth muscle cells which are linked to a greater proliferative and abnormal vasomotor capability. Clinically, applications of vigorous anti-HC regimens perioperatively may be beneficial in maintaining patency over the longer term.
Animals↗
Reduction of experimental vein graft intimal hyperplasia by ketanserin.
Intimal hyperplasia is considered to be the result of smooth muscle cell proliferation. Experimental vein grafts (VG) in the rabbit develop intimal hyperplasia and a contractile response to serotonin (5-HT), mediated by a 5-HT2 receptor. 5-HT is known to stimulate smooth muscle cell proliferation in vitro and therefore may be linked to the development of intimal hyperplasia. This study examines the effect of a 5-HT2 antagonist, ketanserin (KT) on vein graft morphology and function at 14 and 28 days after grafting. Forty-three New Zealand White rabbits underwent common carotid interposition bypass grafting. Twenty-two were treated with KT (0.86 mg/kg/day po) 5 days prior to surgery and thereafter until harvest. The remaining 21 animals acted as controls. VG were harvested at 14 (VG14) and 28 (VG28) days for histology or vasoreactivity. Twenty-three VG were harvested by pressure fixation and the midportions of the grafts were examined by videomorphometry. Standard isometric tension studies in response to serotonin and norepinephrine (NE) were performed on the rings from the remaining 20 VG. Contralateral external jugular veins (CV) from both groups were studied to assess the functional toxicity of KT. There were no toxic effects to KT noted. KT therapy did not affect the functional activity of the CV at 14 or 28 days when compared to controls. When compared to controls, KT significantly reduced the intimal thickness (49 +/- 11 vs 113 +/- 24 microns; P = 0.04) and there was an increase in luminal area (16.89 +/- 2.13 vs 8.41 +/- 1.59 mm2; P < 0.02) in VG28 only.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Temporal thrombocytopenia after engraftment with defined stem cells with long-term marrow reconstituting activity.
Delayed platelet recovery and altered marrow megakaryocytopoiesis was observed in highly irradiated mice following transplantation with 1000 lineage-negative, stem cell antigen-positive (Lin-Sca-1+) cells. Thirty days after transplantation, the mice were thrombocytopenic. Normal platelet levels were reestablished by 90 days after transplantation. Platelet levels were established from the bone marrow with altered megakaryocytopoiesis, however. Megakaryocyte progenitor numbers were found at reduced levels in the reconstituted marrow at all time points assessed. Bone marrow function was also different in that marrow reserve was also diminished. While the transplantation regime did give long-term reconstitution of blood cells, the quality of the marrow reserve was significantly impaired as revealed by response to further hematopoietic challenge. The data indicate that mice transplanted with a population of highly defined stem cells have perturbed marrow function, one characteristic being an altered process of megakaryocytopoiesis.
Animals↗
Molecular mimicry by major histocompatibility complex molecules and peptides accounts for some alloresponses.
One explanation offered for the uniquely high precursor frequencies of T cells which recognize allogeneic major histocompatibility complex (MHC) molecules, and their lack of self-MHC restriction, is that the alloreactive cells are polyclonal populations the primary specificity of which is self-MHC plus peptide X1, X2, ... Xn. These are postulated to cross-react with allo-MHC plus peptides Y1, Y2, ... Yn. It has been further suggested that the structural basis for the crossreactivity between different MHC alleles is the similarity in amino acid sequence of that part of the molecule predicted to make contact with the T cell receptor (TcR). In order to test this concept, T cells were obtained with dual specificity for influenza haemagglutinin (HA), restricted by HLA-DR1Dw1, and for DR4Dw4/Dw14 expressed on allogeneic human B cell lines, and the specificity of one clone was studied in detail. The exposed, TcR-contacting surfaces of these two DR molecules are predicted to be identical. Although the HA-specific response was stimulated by DR1-expressing mouse DAP.3 transfectants, DAP.3 cells expressing the alloantigen DR4Dw4 were unable to stimulate, possibly because of a failure to present the necessary human peptide for anti-DR4 allorecognition. Therefore, the effects of pulsing the DR4Dw4-expressing DAP.3 cells with the HA peptide were examined. This peptide is known to bind to both DR1 and DR4. Addition of the HA peptide restored the anti-DR4Dw4 response. These data support the concept that allorecognition in some responder/stimulator combinations can be explained by cross-reactivity at the level of the MHC molecule and the peptide.
Amino Acid Sequence↗
Structural analysis of human anti-mouse H-2E xenorecognition: T cell receptor bias and impaired CD4 interaction contribute to weak xenoresponses.
T lymphocyte responses to the MHC of an evolutionarily distant species are known to be weak compared with responses against allogeneic MHC products within a species. This fact was used to examine the regions of human MHC class II molecules required for the stimulation of strong primary immune responses against MHC alloantigens. A panel of mouse DAP.3 transfectants expressing the products of wild-type and recombinant DR1/H-2Ek MHC class II genes paired to either DR alpha or H-2E alpha genes was generated, and tested as stimulator cells for purified human T cells. A strong proliferative response to DAP.3 transfectants expressing allogeneic HLA-DR molecules was seen. In contrast, weak or absent responses were recorded against DAP.3 cells expressing H-2E molecules. Substitution of the DR1 beta chain with H-2E beta k led to a dramatic loss of recognition; alpha chain substitution had a less marked effect. Furthermore, replacement of the beta 2 domain of DR1 with H-2E sequence caused 90% inhibition, whereas introduction of the beta 2 domain of DR1 into H-2Ek led to a 10-fold increase in T cell response. These results are most readily explained if the beta 2 domain contributes to the interaction site for the CD4 molecule. Substitution of either half of the beta 1 domain led to a marked loss of response. This was more impressive following substitution of the TCR-contacting alpha-helical region of the domain.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Structural analysis of anti-DR1 allorecognition by using DR1/H-2Ek hybrid molecules. Influence of the beta 2-domain correlates with CD4 dependence.
A segmental analysis of the key regions of HLA-DR1 that control T cell allorecognition was performed by using a series of transfected cell lines expressing the products of recombinant DRB/H-2Eb genes, paired with either DR alpha or H-2E alpha. Four of eight human T cell clones tolerated substitution of the H-2E alpha chain, but only one clone showed any response to the DR alpha/H-2E beta k dimer. Both the membrane-proximal and the membrane-distal domains of the beta-chain played an important part in stimulating these clones. The response of four of eight clones was markedly inhibited by substitution of the H-2E beta 2 for the DR beta 2 domain. This inhibition showed a complete correlation with the sensitivity of the clones to inhibition by anti-CD4 mAb. Taken together, these results suggest that the interaction site for CD4 may include residues on the beta 2-domain. Introduction of H-2Ek sequence into either half of the beta 1-domain led to a complete loss of response by all but two of the clones. This is consistent with these clones having dual specificity for exposed DR1-specific polymorphisms and for DR1-bound peptides. The pattern of response of one of the clones suggested that indirect conformational effects on the alpha 1-domain may also contribute to the influence of the amino-terminal half of the beta 1-domain on T cell recognition. In the presence of H-2E alpha, this clone responded more strongly when the amino-terminal half of the beta 1-domain was of H-2Ek rather than DR1 sequence. This implies that species matching of the floor of the beta 1-domain with the alpha-chain is more important than the presence of the alpha-chain of the parental species.
Amino Acid Sequence↗
The specificity of alloreactive T cells is determined by MHC polymorphisms which contact the T cell receptor and which influence peptide binding.
The separate contributions to allorecognition of peptide-binding and T cell receptor-contacting residues of an allogeneic HLA-DR molecule were investigated by site-directed mutagenesis. Alloreactive T cell clones were generated from a combination of responder (DR1Dw1,DR4Dw14) and stimulator (DR1Dw1, DR4Dw10) whose DR products differed at only three amino acid positions, two of which are predicted to interact with the T cell receptor (67 and 70), and one with bound peptide (71). Transfected murine DAP.3 cells expressing the wild type and mutated forms of DR4Dw10 in which the codons for residues 70 and/or 71 had been altered towards DR4Dw14 were used to stimulate a panel of anti-DR4Dw10 T cell clones. Substitutions at either position 70 or 71, or the combination of the two, led to loss of recognition by the alloreactive T cell clones. This implies that residues involved in peptide binding and residues involved in interaction with the T cell receptor are important for this panel of alloreactive T cell clones. The specificity of these alloreactive T cells for exposed polymorphic residues on the allogeneic MHC molecule was further demonstrated by the inhibitory effects of synthetic peptides, derived from the alpha-helix of the beta 1 domain of the DR4Dw10 molecule.
Animals↗
Production of a cytotoxic human monoclonal antibody with specificity for HLA-DR4 and -DRw10 by cells derived from a highly sensitized kidney recipient.
A human monoclonal antibody which reacts preferentially with HLA-DR4 and -DRw10 B-cell targets has been produced. A human B-cell line, secreting antibody which reacted preferentially with DR4 and DR1 targets, was derived from a highly sensitized kidney recipient who had rejected two grafts. This line was fused with the mouse myeloma P3X63Ag8.653 and a selected hybridoma cloned. The clones secrete IgM(lambda), which reacts strongly with HLA-DR4 and -DRw10 and more weakly with -DRw14 and a proportion of -DR1 B cells in cytotoxicity assays. Using B-cell lines as targets in cytotoxicity and enzyme-linked immunosorbent assays, the antibody gives a broader pattern of reaction, reacting with HLA-DR1, -DR4, -DR9, -DRw10, -DRw14, and some -DR2 targets. The antibody (NI) is currently in use as a reagent for tissue typing.
Animals↗
Canadian therapists' priorities for clinical research: a Delphi study.
Clinically relevant physical therapy research questions were developed by a Delphi technique among 55 teaching hospital physical therapists. The Delphi technique used in this study involved three rounds of questionnaires that included characteristics of anonymity, feedback, ranking with statistical scoring, and use of informed respondents. Fifty-eight initial research questions were narrowed to 11 according to their potential benefit for the patient, for the practice of physical therapy, and for decreasing health care costs. A literature review revealed that each of the 11 questions were as yet unanswered. The use of the survey results to guide and plan for clinical research in physical therapy is discussed.
Canada↗
Functional evidence for the recognition of endogenous peptides by autoreactive T cell clones.
The fine specificity of two human T cell clones responding to autologous HLA-DR1 expressing antigen-presenting cells (APC) in the absence of nominal antigen has been investigated using Epstein-Barr virus-transformed B cells (BCL) of known DR beta 1 domain sequence. It was found that responsiveness was markedly affected by changes in a limited number of residues in this domain. Substitution of the DR1 beta sequence at one residue, position 74, even conservatively, was found to be particularly significant. Located on the beta 1 domain alpha-helix, this residue is predicted to point into the antigen-binding groove and is therefore unlikely to make contact with the T cell receptor. This finding suggests that these T cells are specific for a bound endogenous peptide within the autologous major histocompatibility (MHC) binding groove. The autospecific T cell clones also responded to murine L cell transfectants expressing DR alpha DR1 beta as well as to transfectants expressing the mouse/human hybrid MHC molecule I-E alpha DR1 beta but not to the reciprocal combination DR alpha I-E beta, thus confirming the importance of the beta 1 domain to T cell recognition. In contrast to the autocytotoxicity observed with BCL, cytolysis of the murine L cells expressing the HLA-DR1 molecule was slight and only found at high effector-target ratios. In addition, although fixation enhanced the recognition of BCL, capacity of the murine L cells bearing the HLA-DR1 molecule to stimulate T cell clone proliferation was markedly reduced by aldehyde fixation. When taken together, these results suggest that the endogenous peptides recognized by these autoreactive T cells are of human origin.
Amino Acid Sequence↗
Bipolar fixation of fractures of the distal end of the radius: a comparative study.
This paper reports a prospective study of the treatment of Colles' fractures, either by bipolar fixation or a forearm cast. Bipolar fixation reduced the degree of secondary displacement and no patient required a repeat manipulation. The functional results at 6 months, as assessed by the Scheck system, showed a strong trend in favour of bipolar fixation, but did not reach 95 per cent confidence limits. The technique of bipolar fixation proved to be safe and suitable for use in a busy district general hospital, without the necessity of elaborate equipment.
Bone Nails↗