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Biomedical subjects

T Lund

Publications and source records attributed to T Lund.

At least 91 records · Page 5Linked to original sources
PubMed1996

Agalactosyl IgG and beta-1,4-galactosyltransferase gene expression in rheumatoid arthritis patients and in the arthritis-prone MRL lpr/lpr mouse.

Reduced galactosylation of immunoglobulin G (IgG) is well documented in rheumatoid arthritis (RA), but the reason for this defect is still unknown. There is some evidence supporting a defect in the biosynthetic pathway, and a reduction in the level of beta-1,4-galactosyltransferase (beta-1,4-GalTase) enzyme activity. Since glycosyltransferases are, in general, regulated at the level of transcription, we have measured the level of beta-1,4-GalTase gene expression in B cells from patients with RA and normal control individuals. We found no significant difference in mRNA levels for the transferase in these two groups (P > 0.7). MRL/Mp-lpr/lpr (MRL-lpr) mice develop a spontaneous arthritis with increased levels of agalactosyl IgG (G0). In spite of a significant reduction in the level of beta-1,4-GalTase mRNA in total spleen lymphocytes from MRL-lpr mice compared with the congenic MRL/Mp-(+/+) (MRL-(+/+) mice and with CBA/Ca mice, we found comparable levels of the beta-1,4-GalTase mRNA in purified B cells from both spleen and lymph nodes of the three strains. Amongst the lymphoid compartments examined, the spleen and peripheral blood were found to be the major contributors of G0 in MRL-lpr mice. These data indicate that in neither human RA, nor in an animal model of this disease, is reduced IgG galactosylation caused by impaired expression of the beta-1,4-GalTase gene in B lymphocytes. Furthermore, splenic B cells, which have normal levels of beta-1,4-GalTase mRNA, appear to be a major source of G0 in MRL-lpr mice.

Animals↗
PubMed1996

Coexpression of H2-Mb and H2-Ab genes during fetal and postnatal development.

The major histocompatibility complex (MHC) class II-like molecules, H2-M, have an essential role in processing and presentation of antigens by the MHC class II molecules, because functional inactivation of these genes lead to surface expression of MHC class II molecules devoid of associated foreign peptides. We have used in situ hybridization to examine the expression of MHC class II and H2-Mb genes in embryonic and neonatal mice and show here that expression of H2-Ab and H2-Mb mRNA is absent in 13.5-day-old mice. However, mRNA for both genes could be detected in the thymus of 14.5- and 15.5-day-old embryos, and the patterns of hybridization suggested that the two genes were expressed in the same cells. In spleen and thymus of neonatal mice the H2-Ab and H2-Mb genes were also coexpressed, with expression being localized to the white pulp of the spleen and to the thymic medulla, which are rich in antigen-presenting cells. The steady-state levels of H2-Ab mRNA appeared to be approximately 10 to 14 times greater that the level of H2-Mb mRNA molecules, irrespectively of the tissue and age, reflecting the different functions of the two molecules.

Animals↗
PubMed1996

Enhancement in antigen binding by a combination of synergy and antibody capture.

The effects of orientating pairs of synergistic monoclonal antibodies (mAb) on binding of human chorionic gonadotropin (hCG) was studied by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Antibody synergy towards hCG required two functionally intact antibodies located adjacent to each other and with different epitope specificities. We investigated whether immobilization procedures avoiding protein denaturation, increasing proper orientation and promoting higher molecular flexibility of the synergistic mAb resulted in significantly enhanced antigen, binding. Synergistic mAb pairs captured through their Fc-region by protein G or a polyclonal serum against the Fc-part of mouse IgG could be used at 10-fold lower coating concentrations to achieve maximal binding of the analyte as compared with the same mAb pairs coated directly onto polystyrene. The synergistic effect observed with protein A used as capture varied greatly with the subclasses of the two synergistic antibodies employed. Scatchard analysis revealed that the number of functionally synergistic antibody sites participating in the binding of hCG for one mAb pair was about 10 times higher for the protein G-captured as compared with the directly coated synergistic pair. Biotinylated synergistic mAb pairs, coated directly or captured by streptavidin, did not display any enhanced antigen binding when tested in SPR or ELISA. With SPR, synergy was only observed when the synergistic mAb had been captured through their Fc-region. Using protein G or a polyclonal rabbit anti-IgG1 serum as capture reagents in SPR, synergistic triple mAb combinations against hCG were demonstrated.

Animals↗
PubMed1996

A herpesvirus saimiri membrane protein required for interleukin-2 independence forms a stable complex with p56lck.

ORF-2, a 32-kDa viral protein expressed by herpesvirus saimiri-transformed lymphocytes, is essential for transformation and is expressed on the plasma membrane of transformed cells. The current work now shows that most (approximately 80%) of ORF-2 resides in the cytoplasm, while only a small portion protrudes from the cell surface. Expressed as a glutathione S-transferase fusion protein, ORF-2 was found to interact with a 56-kDa cellular protein in untransformed, herpesvirus saimiri-transformed, and Jurkat lymphocytes. Microsequencing proved that this protein is the lymphocyte-specific tyrosine protein kinase p56lck. Two regions of ORF-2 were found to be required for p56lck interaction. Current evidence suggests that the interaction of ORF-2 with p56lck plays a key role in the specific transformation of T lymphocytes to an interleukin-2-independent phenotype.

Amino Acid Sequence↗
PubMed1995

Aspartate at position 57 of nonobese diabetic I-Ag7 beta-chain diminishes the spontaneous incidence of insulin-dependent diabetes mellitus.

MHC class II genes have been shown to influence the development of the autoimmune disease insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse. In human IDDM it has been suggested that the presence of an aspartate at position 57 of the DQ beta-chain might be important in determining resistance to development of IDDM. The involvement of MHC class II genes in IDDM was investigated through the introduction of MHC encoding transgenes. We show that introduction of a mutated I-Ag7 Ab gene which encodes an aspartate at position 57 reduces the incidence of IDDM but does not prevent insulitis, sialadenitis, or the development of insulin and nuclear autoantibodies.

Amino Acid Sequence↗
PubMed1995

A polycistronic transcript in transformed cells encodes the dihydrofolate reductase of herpesvirus saimiri.

Herpesvirus saimiri, an oncogenic gamma herpesvirus of primates, is the only eukaryotic virus that carries the entire metabolic gene set for a complex biochemical synthesis. Every element of the thymidine synthesis gene cascade is present in the virus, and their function is probably related to the uniquely high A + T content of the genome. Although one member of the gene set, dihydrofolate reductase (DHFR), is mapped in a region required for oncogenesis, very little is known of the expression and function of this gene in transformed cells. We report the expression of the DHFR sequence on a novel, unique tricistronic transcript in virally transformed tumor cells. The DHFR sequence is the first open reading frame on a 5.3 kb minor transcript. Alpha-amanitine sensitivity indicates that it is an RNA polymerase II transcript, and since it is also polyadenylated it appears to be a functional, relatively unstable (half-life 3 hr) mRNA. Initiation of transcription uniquely overlaps with the HSUR3 small RNA gene. Expression of the small transcript appears to be alpha-amanitine resistant, implicating polymerase III transcription. Together with the remarkably low-level expression of HSUR3 in tumor cells, the data may indicate transcription interference between two different RNA polymerases, with unusual overlapping regulation and initiation.

Base Sequence↗
PubMed1995

A model of fluid resuscitation following burn injury: formulation and parameter estimation.

A dynamic compartmental model is developed to describe the redistribution of fluid and albumin between the circulation and the intact and injured interstitia following burn injury in humans. Transcapillary fluid and albumin exchange is described by a coupled Starling mechanism, while the effect of the burn is represented by time-dependent perturbations to all three compartments. The unknown model parameters are determined for two groups of patients, having less than and greater than 25% total body surface area burns, by statistical fitting of model predictions to patient data from two sources. The parameters include the perturbations to the fluid filtration coefficients in uninjured and injured tissue, GkF,Tl and GkF,BT, respectively, the relaxation coefficient, r, which describes the exponential decay of the perturbations, and the exudation factor, EXFAC, which relates the protein concentration in the exudate to that in the injured tissue. Perturbations to other parameters, including the membrane permeability-surface area product and the albumin reflection coefficient in the injured and uninjured tissues, are determined based on interrelationships with GkF,Tl and GkF,BT. The values of GkF,BT, when corrected for tissue destruction and decreased post-injury perfusion, are in reasonable agreement with the limited experimental data available from the literature. The model and its parameters are further validated by comparing the simulated patient responses to the clinical data used in the parameter estimation as well as to data available from two additional sources.

Body Fluid Compartments↗
PubMed1995

Regulation of human growth hormone-binding protein production by human growth hormone in a hepatoma cell line.

The mechanism by which growth hormone-binding protein (GH-BP) is generated in humans remains unclear. To address this question, we analysed human GH-receptor/GH-BP gene expression in a human hepatoma cell line (HuH7). Northern hybridisation showed that HuH7 cells contain a single mRNA species hybridising with a probe for the sequences encoding the extracellular domain of the hGH-receptor/GH-BP. These data were confirmed by solution hybridisation methods. Thereafter, the cells were treated with r-hGH at physiological (12.5, 25, 50 ng/ml) and supra-physiological (150, 500 ng/ml) concentrations over the period of 48 h. At intervals, RNase protection assays were performed to determine GH-receptor/GH-BP mRNA levels, nuclear run-on assays were carried out to determine whether changes in mRNA levels represented changes in transcription rate, and a radio-ligand binding assay was performed to measure levels of GH-BP in the medium. We found that the r-hGH-regulated changes in GH-receptor/GH-BP mRNA levels detected with the probe for sequences encoding the extracellular domain of human GH-receptor/GH-BP were identical to those previously detected using a probe for the sequences encoding the transmembrane/intracellular domain of the human GH-receptor. In addition, we found that r-hGH had a rapid effect on the levels of GH-BP in the culture medium, which differed from its effect on the GH-receptor/GH-BP mRNA levels. Furthermore, lowering of temperature resulted in a decrease of GH-BP released into the medium implying that enzymes may be involved in the releasing mechanism. These data support the idea that GH-receptor and GH-BP are encoded by a single mRNA species in humans. In addition, they suggest that GH-BP levels are not an accurate reflection of GH-receptor/GH-BP mRNA levels, but that GH-BP production is subject to r-hGH-dependent post-transcriptional regulation, perhaps at the level of post-translational cleavage of the full-length GH-receptor protein. The notion that GH-BP measurements might represent GH-receptor status at the functional level must therefore be taken with caution.

Blotting, Northern↗
PubMed1995

A herpesvirus saimiri protein required for interleukin-2 independence is associated with membranes of transformed T cells.

A region of the herpesvirus saimiri genome encoding an mRNA with two open reading frames (ORFs) has been identified to be essential for transformation of T cells. Deletion of either ORF resulted in the loss of transforming ability. ORF-1 has been shown to code for a collagen-like oncoprotein. This study shows for the first time that the bicistronic mRNA can translate a 32-kDa protein from ORF-2. Polyclonal serum to ORF-2 was generated by using a glutathione fusion protein. Using this antiserum, ORF-2 was localized in cell membranes and is expressed on the outer cell membrane. The half-life of this membrane protein was found to be about 5.5 h. Limited sequence similarity was found between ORF-2 and interleukin-11; however, no secretion of ORF-2 protein was detected in supernatants from transformed cells. Further studies are required to investigate the potential interaction with the interleukin-11 receptor.

Animals↗
PubMed1994

Physical mapping of the rat MHC class II genes shows a high level of interspecies conservation.

We report here a pulsed-field gel electrophoresis map of the rat major histocompatibility complex (MHC) class II region. Using probes for the recently discovered Tap-1 and Tap-2 genes and the different MHC class II genes, we found the gene order in the rat MHC (RT1) region to be RT1.H-Tap-1-Tap-2-Bb-Ba-Db-Da. Moreover, the distance between the Tap-1 and the RT1.Da genes is approximately 150 kb. This, together with recent mapping of the RT1 class II region, demonstrates an extensive colinearity in the MHC region of different species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Pulsed field gel electrophoretic analysis of the rat major histocompatibility complex class III region shows extensive inter-species conservation.

Using pulsed field gel electrophoresis (PFGE), we have examined the rat major histocompatibility complex (MHC) for the presence of a number of new class III region genes recently identified in the human MHC. We find homologous genes to the human G1, G2, G4, G7a, G9, G9a, G10, G13, G15, and G18 genes, but not the G8 gene in the rat genome, and show that these are linked to the rat TNF-alpha and C4/Slp loci. A long-range restriction map has been constructed on the basis of a PFGE analysis which demonstrates extensive co-linearity in the positions of the homologous sequences in the region between the C4/Slp and TNF loci in the rat MHC when compared with that of the human MHC class III region.

Animals↗
PubMed1994

Alpha-Trinositol inhibits edema generation and albumin extravasation in thermally injured skin.

Pharmacologic attempts to reduce edema generation and albumin extravasation into thermally injured skin have until recently been disappointing unless the drugs (usually antiphlogistic or anti-inflammatory drugs) were given before injury. We have studied the effect of alpha-trinositol (PP56, i.e., 1D-myo-inositol-1,2,6-trisphosphate) given after the injury in an experimental full-thickness 10% TBSA scald burn in anesthetized rats. Total tissue water content (TTW) and albumin extravasation (Ealb) were determined in injured and noninjured skin (series I, n = 12). Interstitial fluid hydrostatic pressure (Pif) was measured in injured skin (series II, n = 14). alpha-Trinositol was administered (alpha-trinositol groups) as an i.v. bolus (40 mg/kg) at 5 minutes after injury followed by an i.v. infusion (1.3 mg/kg/min). In both series a placebo group received burn injury and normal saline in equal volumes instead of alpha-trinositol. Compared with placebo, alpha-trinositol reduced TTW and Ealb as well as the increased negatively of Pif in injured tissue significantly. The effect on Ealb was most prominent, with a reduction from 153.9 +/- 35.6 (SEM) microL/g in the NaCl group to 23.1 +/- 6.3 after alpha-trinositol (p < 0.005). Total tissue water was reduced from 2.51 +/- 0.13 to 2.17 +/- 0.06 mL/g (p < 0.05) and Pif (measured between 21 and 40 minutes postinjury) from -24.7 +/- 4.1 to -3.2 +/- 1.1 mm Hg (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1994

Studies on the thymus of non-obese diabetic (NOD) mice: effect of transgene expression.

The non-obese diabetic (NOD) mouse is a good model of insulin-dependent diabetes mellitus. Autoreactive T cells may play a fundamental role in disease initiation in this model, while disregulation of such cells may result from an abnormal thymic microenvironment. Diabetes is prevented in NOD mice by direct introduction of an E alpha d transgene (NOD-E) or a modified I-A beta chain of NOD origin (NOD-PRO or NOD-ASP). To investigate if disease pathology in NOD mice, protection from disease in transgenic NOD-E and NOD-PRO and partial protection from disease in NOD-ASP can be attributed to alterations in the thymic microenvironment, immunohistochemical and flow cytometric analysis of the thymi of these mouse strains was studied. Thymi from NOD and NOD-E mice showed a progressive increase in thymic B-cell percentage from 12 weeks of age. This was accompanied by a concomitant loss in thymic epithelial cells with the appearance of large epithelial-free areas mainly at the corticomedullary junction, which increased in size and number with age and contained the B-cell clusters. Such thymic B cells did not express CD5 and were absent in CBA, NOD-ASP and NOD-PRO mice as were the epithelial cell-free spaces, even at 5 months of age. Therefore the mechanisms of disease protection in the transgenic NOD-E and NOD-ASP/NOD-PRO mice may differ if these thymic abnormalities are related to disease.

Animals↗
PubMed1994

Reduced galactosyltransferase mRNA levels are associated with the agalactosyl IgG found in arthritis-prone MRL-lpr/lpr strain mice.

MRL-lpr/lpr strain mice have defectively glycosylated IgG. This may be related to the rheumatoid arthritis (RA)-like disease that occurs in these mice, because a similar glycosylation defect is seen in human subjects with RA. Whilst it is known that this defect is associated with reduced activity of the beta-1,4-galactosyltransferase (beta-1,4-GalTase) enzyme, the cause of this reduced activity is at present unknown. We have therefore examined the molecular genetics of beta-1,4-GalTase in MRL-lpr/lpr mice. Using 10 different restriction endonucleases we found no evidence for a polymorphic variant of the gene in glycosylation-defective mice. However, the level of mRNA for beta-1,4-GalTase was lowest in the MRL-lpr/lpr mice, the strain with the most poorly galactosylated IgG of the four strains examined. Thus, the reduced level of IgG oligosaccharide galactosylation found in MRL-lpr/lpr strain mice appears to be related to either an altered transcriptional level of, or altered mRNA stability for, beta-1,4-GalTase in lymphocytes from these mice.

Animals↗
PubMed1994

[Handling of organ donors].

Patients suffering from complete destruction of the brain and brain stem are the most common source of organs for transplantation purposes. However, as the lack of such organs is currently the most limiting factor regarding the scope of transplantation treatment, it is of the utmost importance that all potential donor organs are well maintained.

Brain Death↗