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Biomedical subjects

W H Marshall

Publications and source records attributed to W H Marshall.

At least 19 recordsLinked to original sources
PubMed2000

Beryllium presentation to CD4+ T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease.

Chronic beryllium disease results from beryllium exposure in the workplace and is characterized by CD4(+) T cell-mediated inflammation in the lung. Susceptibility to this disease is associated with particular HLA-DP alleles. We isolated beryllium-specific T cell lines from the lungs of affected patients. These CD4(+) T cell lines specifically responded to beryllium in culture in the presence of antigen-presenting cells that expressed class II MHC molecules HLA-DR, -DQ, and -DP. The response to beryllium was nearly completely and selectively blocked by mAb to HLA-DP. Additional studies showed that only certain HLA-DP alleles allowed presentation of beryllium. Overall, the DP alleles that presented beryllium to disease-specific T cell lines match those implicated in disease susceptibility, providing a mechanism for this association. Based on amino acid residues shared by these restricting and susceptibility DP alleles, our results provide insight into the residues of the DP beta-chain required for beryllium presentation.

Alleles↗
PubMed1999

Parentage testing implications of male fertility after allogeneic bone marrow transplantation.

Fertility is expected to be reduced after the extensive chemotherapy and/or radiotherapy that is needed for conditioning prior to bone marrow transplantation. However, a male patient can be fertile, and in very rare situations such as reported here, this may confuse subsequent paternity testing. The patient, initially excluded as the biological father by red cell types but not by HLA, was subsequently included after the history of his previous marrow transplant was revealed, a review of the HLA results and further RFLP testing on buccal mucosal cells. This case points to the need for good history taking before performing paternity testing.

Adult↗
PubMed1998

HLA-DP epitope typing using monoclonal antibodies.

We have made a panel of murine anti-DP monoclonal antibodies for serological typing of HLA-DP polymorphisms; they can be used in microcytotoxicity (for 7 epitopes) and binding assays (for 8 epitopes). The antibodies detect polymorphic differences in both alpha and beta chains. As immunogens we sometimes used B-lymphoblastoid lines or purified DP molecules but mostly used mouse fibroblast transfectants expressing DP molecules. The DP beta genes were made from a cloned DPB1*0201 gene by replacing its major area of polymorphism with matching stretches of DNA amplified from other alleles; cloned DPA1*01 and DPA1*02 genes were used for transfection along with the beta chain genes. The monoclonal antibodies showed reaction patterns that correlated with the presence of particular amino-acid sequence motifs; thus none of the antibodies is allele-specific. They bind instead to epitopes which are found on a number of different HLA-DP types. We have constructed frequency tables so that the epitope (motif) data can be interpreted as the most likely genotype in each case. The basic assumption to justify this work is that HLA-DP matching or mismatching will likely influence transplant outcome, particularly in bone marrow transplantation. The present challenge is to define permissive and nonpermissive combinations of HLA-DP; it may be that matching for epitopes, rather than for full alleles, will help to resolve this issue.

Animals↗
PubMed1997

Undifferentiated (embryonal) sarcoma of the liver: pathologic basis of imaging findings in 28 cases.

PURPOSE: To correlate the imaging and pathologic features of undifferentiated (embryonal) sarcoma (UES) and account for the discrepancy between the solid appearance at ultrasound (US) and the almost cystlike appearance at computed tomography (CT) and magnetic resonance (MR) imaging. MATERIALS AND METHODS: The clinical, pathologic, and imaging findings in 28 patients (age range, 3-49 years) with pathologically proved UES were retrospectively reviewed. All patients underwent at least one cross-sectional imaging study to include CT (27 patients), US (21 patients), and MR imaging (six patients). Tumor size, gross morphology (n = 27), histologic features, and proportion of solid and cystlike components were evaluated and correlated to the imaging findings. RESULTS: The mean transverse diameter of the tumors was 14 cm (range, 10-25 cm). At gross examination, the tumors were predominantly solid (mean, 83% of tumor volume), and pathologic and US findings were concordant. Conversely, CT scans showed low attenuation (approximately that of water) in 88% of the tumor volume and T2-weighted MR images showed high signal intensity (approximately equal to that of cerebrospinal fluid) in 89% of the tumor volume. CONCLUSION: UES shows a misleading cystlike appearance at CT and MR imaging compared with US and pathologic findings. In a child or young adult with a liver tumor, this finding is useful in making a prospective diagnosis and avoiding misguided attempts at drainage.

Adolescent↗
PubMed1997

Imaging of pediatric liver tumors.

Primary pediatric liver tumors are a unique group of neoplasms. The age at presentation, clinical sigma symptoms, and imaging characteristics provide helpful clues in making a prospective diagnosis and in tailoring a differential diagnosis. The role of imaging is fourfold: characterization, localization, determination of resectability, and follow-up use. MR is usually complementary to ultrasound and CT.

Bile Duct Neoplasms↗
PubMed1995

HLA-DR residues accessible under the peptide-binding groove contribute to polymorphic antibody epitopes.

Many residues involved in polymorphic antibody-binding epitopes on class II molecules are located on the alpha-helix of DR beta chains. Although they have received less attention, residues in the peptide-binding groove and second domain of the DR beta chain may also be critical for polymorphic anti-DR antibody epitopes. In this study, we used transfectants expressing site-directed mutations at positions in the HLA-DR beta 1 and beta 2 domains and flow cytometry to define the epitopes of several polymorphic anti-DR antibodies. Both DR(beta 1*0403) residues 14 and 25 were shown to be involved in the epitopes of mAbs DA6. 164, HU-20, Q5/6, and 50D6, and DR(beta 1*0701) residue 14 was shown to be critical for the epitopes of two DR7-specific mAbs, SFR 16-DR7M and TAL13.1. Unlike most other residues shown to be important in antibody-binding epitopes, residue 14 is located in the floor of the peptide-binding groove and residue 25 is in an outer loop, each with their side chains pointing down, such that antibodies may directly contact these residues from below the binding groove. Two residues in the beta 2 domain, beta 180 and beta 181, were also shown to be involved in the epitopes of three polymorphic anti-DR mAbs, NFLD.D1, NFLD.M1, and LY9. Although these two residues are close to the transmembrane domain in the linear sequence, their solvent accessibility in the DR1 structures is quite impressive. Our data provide new evidence that residues accessible under the peptide-binding groove contribute to polymorphic antibody-binding epitopes.

Amino Acid Sequence↗
PubMed1995

Influence on antibody recognition of amino acid substitutions in the cleft of HLA-DQ2 molecules. Suggestive evidence of peptide-dependent epitopes.

The purpose of this study was to identify polymorphic residues of DQ2 beta chains which are involved in the epitopes recognized by DQ2-reactive mAbs. Binding studies were performed on a panel of DQ2 mutant cells made by transfection of a DQ (alpha 1*0501, beta 1*0301)-positive B-LCL with DQB1 genes encoding either wild-type or mutated DQ beta 1 *0202. Several aa substitutions were found to influence the binding of DQ2 mAbs. All but one of the mAbs were clearly sensitive to the introduced aa substitutions, but individual mAbs were differentially influenced, suggesting that they recognized different epitopes on the DQ2 molecule. Substitutions in positions 30 and 37 of the peptide-binding cleft were also found to influence the binding of some mAbs. Second, two mAbs, NFLD.M71 and NFLD.M102, which bound to DQ(alpha 1*0501, beta 1*0202) expressed in human cells and were sensitive to the introduction of aa substitutions in the cleft, bound weakly to the DQ(alpha 1*0501, beta 1*0202) heterodimer when expressed in a transfected murine NIH 3T3 cell line. Together this indicates that some DQ2-reactive mAbs may recognize peptide-dependent epitopes.

3T3 Cells↗
PubMed1995

Identification of woodchuck class I MHC antigens using monoclonal antibodies.

Two class I major histocompatibility complex (MHC) proteins with molecular masses of 43- and 39-kDa were identified in the cell surface membranes of normal woodchucks using a newly developed antiwoodchuck class I monoclonal antibody (mAb) B1b.B9 and immunoblotting. B1b.B9 was generated by immunizing mice with viable woodchuck peripheral blood mononuclear cells and was selected for anti-class I MHC reactivity using a cellular enzyme-linked immunoassay, indirect immunofluorescence on tissue sections and flow cytofluorimetry. The distribution pattern of class I MHC antigen on woodchuck lymphoid cells was found to be similar to that reported in other species. Also, the antigen expression on normal woodchuck hepatocytes was comparable to that observed on normal human liver parenchymal cells; thus, the antigen was not detected on hepatocytes by staining of liver tissue sections, but was found by indirect immunofluorescence staining of isolated liver cells. Western blot analysis of the plasma membranes from normal woodchuck hepatocytes revealed the presence of a single species of class I MHC heavy chain protein with a molecular mass of 43-kDa, whereas splenocyte plasma membranes showed intense expression of a 43-kDa species, as well as the presence of a 39-kDa protein. The 39- and 43-kDa proteins were extracted with Triton X-114 to the hydrophobic protein phase, suggesting that they both contain a hydrophobic transmembrane domain. The data obtained indicate that the B1b.B9 identifies a nonpolymorphic epitope of woodchuck class I MHC heavy chains, providing an important reagent for the study of the pathogenesis of hepatitis B virus infection in a woodchuck model.

Animals↗
PubMed1994

Coordinate defects in human histocompatibility leukocyte antigen class II expression and antigen presentation in bare lymphocyte syndrome.

The human immunodeficiency, type II bare lymphocyte syndrome (BLS), has been attributed to a defect in the transcription of class II histocompatibility genes. Immunocompetence, as assessed by functional exogenous antigen presentation, was not restored in immortalized B cells, derived from a BLS patient, after transfection with HLA-DR class II structural genes. Incubation of protein antigens, as well as infectious virus, with DR-transfected BLS cells failed to induce activation of antigen-specific helper T lymphocytes. Peptide antigens were presented by class II molecules displayed on BLS cells, although the conformation of these class II proteins was altered as indicated by epitope mapping. This defect in antigen presentation was independent of the specific class II DR allele transfected into BLS cells. Genetic complementation analysis has been used with BLS cells to demonstrate that the defect in class II gene transcription is linked to the absence of a trans-acting factor. Similarly, functional class II dimers were restored after in vitro fusion of cells derived from two distinct BLS complementation groups, implying that specific transcriptional control elements are shared by a gene critical for antigen presentation and genes encoding HLA class II antigens. Thus, two important functionally linked pathways of class II molecules, structural gene expression and antigen presentation, share a common regulatory pathway defective in BLS.

Alleles↗
PubMed1994

Analysis of monoclonal antibodies specific for unique and shared determinants on HLA-DR4 molecules.

The specificities for seven mAbs to HLA-DR4 were determined initially using homozygous BCLs and L-cell transfectants expressing wild-type DR molecules. Three antibodies (NFLD.D1, NFLD.M1, and NFLD.D7) bound all DR4 molecules, but only one was specific for DR4. Four antibodies (NFLD.D2, NFLD.D3, NFLD.D8, and NFLD.D10) reacted with some but not all DR4 subtypes and had extra reactions, particularly with DR gene products associated with susceptibility to RA. To localize the antibody-binding epitopes on DR4 molecules, the antibodies were then analyzed on transfectants expressing hybrid genes, which were generated by exon shuffling of DRB1*0403 and DRB1*0701. Two of the pan-DR4 antibodies bound epitopes that require the beta 2 domain while the third mapped primarily to the HVR-I region. One antibody NFLD.D10 to subtypes of DR4 mapped to residues 40-97 on DR beta 1*0403 chains. Comparison of reaction patterns with amino acid sequences suggest that the antibodies against subtypes of DR4 are specific primarily for a region containing sequences postulated to determine susceptibility to RA.

Amino Acid Sequence↗
PubMed1994

Amino acids in the peptide-binding groove influence an antibody-defined, disease-associated HLA-DR epitope.

A shared amino-acid sequence on the alpha helix of certain DR beta 1 chains is predicted to generate a 'shared epitope' that is implicated in susceptibility to the development of rheumatoid arthritis (RA). Different relative risks (RR) for disease susceptibility and severity conferred by these DR beta 1 chains suggest that their 'shared epitopes' are not equivalent. A set of monoclonal antibodies (MoAb) that map to the critical region, and for which optimal binding depends on DR context and cell lineage, was used to test this idea. Mapping experiments using mutated DR beta 1* molecules showed that the antibody-binding epitopes are overlapping; residue 70Q is pivotal for each, but neighbouring residues on the alpha helix and on the floor of the groove are also involved. Importantly, these epitopes are profoundly modified by peptide loading of DR beta 1*0401 molecules. These data suggest that 'shared epitopes' on DR molecules that are associated with RA are influenced by their context; such structural modifications may be the basis for the varying susceptibilities conferred by these DR molecules for the development of RA.

Amino Acid Sequence↗
PubMed1992

A low cost microprocessor-controlled electrofusion and electroporation system.

An important recent development in the field of biotechnology has been the use of high electric fields to render cell membranes temporarily permeable (electroporation). Cells in this state are receptive to gene transfer or can be induced to fuse with each other (electrofusion) to form hybrid cells containing the combined characteristics of the parent cells. A major reason for fusing cells is to form hybridoma cells which secrete monoclonal antibodies. A problem for research workers has been the high price of some of the electrofusion equipment. This problem has been addressed by designing a device that is inexpensive (less than $800 Canadian) and can be assembled by an electronics technician. The system uses a Radio Shack CoCo III microcomputer which is programmed in BASIC and controls the electroporation voltage pulse amplitude (25-500 V) and duration (2-275 microseconds) to an electrofusion chamber; this yields electric field strengths of 0.25-5 kV cm-1 for a 0.1 cm electrode spacing. The system is capable of delivering pulse currents up to 3 A. This paper provides technical details on how to construct an instrument that has greater flexibility at less cost than comparable commercially available instruments.

Biotechnology↗
PubMed1992

Increased limb salvage with intraoperative and postoperative ankle level urokinase infusion in acute lower extremity ischemia.

Over a 30-month period (May 1988 to November 1990) 143 acutely ischemic lower extremities (126 patients) were treated with an aggressive surgical approach that included ankle level tibial-peroneal artery thromboembolectomy. Twelve lower extremities in 10 patients that remained ischemic were further treated with adjuvant ankle level urokinase infusion. Sixteen ankle level arteries in 12 extremities were infused with an intraoperative bolus (1 to 2) of urokinase (50,000 to 100,000 units). Continuous postoperative urokinase (25,000 to 50,000 units per catheter per hour x 1 to 5 days) was infused through ankle level arteriotomies in 10 extremities (14 arteries) that did not improve with the initial intraoperative bolus. Concomitant bypass grafting was necessary in four extremities. With adequate inflow established, adjuvant ankle level urokinase salvaged all 12 extremities. The mean increase in ankle/brachial pressure index was 0.84. During continuous postoperative urokinase infusion, lower extremity bleeding requiring blood transfusion occurred in four patients (50%). No deaths occurred in the operative period. Although rhabdomyolysis occurred in 90% of patients, no patients had renal insufficiency. The addition of ankle level urokinase delivery increased the potential limb salvage from 90% of the entire 143 extremities treated during this period to an actual limb salvage of 98%. A mean follow up of 13 months (6 to 36 months) identified one late amputation. Despite the demanding postoperative management required in these patients and the frequent need for early reoperation, the limb salvage obtained justifies this aggressive adjuvant technique in the management of the acutely ischemic lower extremity.

Aged↗
PubMed1991

Transfection of HLA genes using genomic DNA.

Mouse L cells expressing HLA genes are potentially useful for producing and analyzing monoclonal antibodies (mAb) to HLA molecules. This paper describes the preparation of transfectants using uncloned human DNA and three methods to isolate the HLA-expressing transfectants. Transfectant libraries were made by cotransfecting mouse thymidine kinase (tk)-deficient L cells with a calcium phosphate precipitate containing genomic DNA and tk plasmid DNA. Transfectants expressing HLA genes were isolated using these methods: immunomagnetism, replicate-plating combined with cellular enzyme-linked immunoassay, and sorting using a fluorescence-activated cell sorter. Two HLA-A2 transfectant were isolated using immunomagnetism, two HLA-A24 transfectants by replicate plating, and one HLA-Bw60 transfectant by FACS. However, no transfectants were isolated that stably expressed class II genes. The class I transfectants have been useful in characterizing several locally prepared mAbs which bind to monomorphic determinants on class I HLA molecules. Two of the transfectant lines, one expressing HLA-A2 (8001) and the other HLA-A24 (8008), have been included in the collection of lines distributed for use in the Eleventh International Histocompatibility Workshop.

Antibodies, Monoclonal↗
PubMed1991

Carotid endarterectomy in a community hospital surgical practice.

Three hundred twenty-four carotid endarterectomies (CEAs) were performed on 303 patients over 5 years. Sixty per cent of the patients were symptomatic with completed stroke (36.4%), amaurosis fugax (35.4%) or transient ischemic attack (TIA) (50.5%). Some patients had multiple symptoms. Perioperative stroke occurred in four patients (1.2%) and 30-day mortality in five (1.5%). The combined stroke-mortality rate was 2.8 per cent. Other postoperative complications included TIA (1.9%), cranial nerve injury (3.1%), wound hematoma (6.5%), and hypertensive reperfusion syndrome (9.6%). Ten early reoperations were performed for wound hematoma (7) or technical problems (3). Follow-up of 284 CEAs (88%) at a means of 31 months revealed 33 late deaths, with two due to stroke. Late strokes occurred in 11 patients (3.9%). Five late strokes were ipsilateral (1.8%) and six were contralateral (2.1%) to the operated carotid artery. Ninety-seven carotid arteries were evaluated by duplex ultrasound scanning at a mean postoperative interval of 27.2 months. Ninety-two per cent had 0-30 per cent restenosis, 5 per cent had 40 per cent to 60 per cent restenosis and 3 per cent had 70 per cent or greater restenosis. The authors conclude that CEA can be performed with acceptable morbidity and mortality rates and that it is a durable operation that reduces the risk of late stroke.

Adult↗
PubMed1989

Multiple regions of HLA-DR beta 1 chains determine polymorphic epitopes recognized by monoclonal antibodies.

To investigate the locations of antibody binding epitopes on HLA class II molecules, four DR4/7 beta 1 hybrid cDNA were constructed by exchanging the DNA encoding the NH2-terminal portions (amino acids 1 to 40) or the COOH-terminal portions (amino acids 41 to 94) of the first domains of DR4 beta 1- and DR7 beta 1-chains, in association with DNA encoding either the DR4 beta 1 or DR7 beta 1 second domains. Transfectants expressing a DR alpha cDNA and a wild-type DR4 beta 1 or DR7 beta 1 cDNA or one of four hybrid DR4/7 beta 1 cDNA were produced, and the binding to the transfectants of anticlass II mAb, which detect polymorphic epitopes on either DR4 or DR7 molecules, was analyzed. Four different patterns of mAb binding to the transfectants were observed, indicating that multiple regions of DR beta 1-chains play the predominant roles in the contributions of these chains to polymorphic epitopes recognized by mAb on intact molecules. The relevant regions of these chains and the number of mAb that recognize the associated polymorphic epitopes are: 1) the COOH-terminal portion of the first domain of DR4 beta 1; a DR4-specific mAb, 2) the NH2-terminal portion of the first domain of DR7 beta 1; two mAb, including a DR7-specific mAb, 3) the NH2-terminal portion of the first domain of DR4 beta 1; seven mAb, and 4) the second domain of DR4 beta 1; one mAb.

Amino Acid Sequence↗