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Biomedical subjects

G D Wu

Publications and source records attributed to G D Wu.

At least 37 records · Page 2Linked to original sources
PubMed1998

Xenoantibodies to pig endothelium are expressed in germline configuration and share a conserved immunoglobulin VH gene structure with antibodies to common infectious agents.

BACKGROUND: The rejection of pig xenografts in humans is initiated by preformed antibodies that may be related to the natural antibodies that formulate a first line of defense against infectious agents. Immunoglobulin gene variable domains encoding the antibodies that react with similar epitopes expressed on xenoantigens and bacteria may share structurally similar antigen-binding site configurations. METHODS: We sequenced the VH immunoglobulin genes and germline progenitors of two rat monoclonal antibodies that recognize pig xenoantigens. Nucleic and amino acid sequences of these xenoantibodies were compared with immunoglobulin genes encoding antibodies that react with bacteria or viruses. RESULTS AND CONCLUSIONS: VH genes encoding rat anti-pig xenoantibodies are expressed in germline configuration and share structural similarities, including identical amino acids in key antigenic contact sites that define antibody canonical structural groups, with antibodies to infectious agents.

Amino Acid Sequence↗
PubMed1998

Application of free-flow electrophoresis to the purification of trichosanthin from a crude product of acetone fractional precipitation.

The application of free-flow electrophoresis (FFE) to the purification of trichosanthin (TCS) from a crude product of acetone fractional precipitation was investigated. An electrophoresis technique, combining field step electrophoresis (FSE) and zone electrophoresis (ZE) to a one-step procedure, was optimized until a satisfactory purification factor (1.35), high resolution, and purity (>99%) were achieved. Testing several separation buffer systems revealed that a throughput of 14.2 mg/h can be obtained when the very basic TCS (pI 10.1) was dissolved and electrophoresed in a phosphate buffer system of pH 4. The purity of electrophoresed trichosanthin was proved by a variety of analytical methods, such as sodium dodecyl sulfate (SDS)-gel electrophoresis, capillary isoelectric focusing (CIEF), and sequencing of N- and C-termini. The high purity and large throughput achieved at low cost by using FFE indicates that this method can be employed for TCS purification.

Acetone↗
PubMed1998

The effects of interaction between morphine and interleukin-1 on the immune response.

The findings presented here clearly indicate an association between opioid- and cytokine-dependent systems. Exposure to exogenous opioids can produce profound effects on IL-1-mediated immune responses. Chronic exposure to morphine appears to disrupt the brain-immune axis by desensitizing the IL-1 activation of the HPA axis, and consequently potentiate the LEA response to IL-1. On the other hand, endogeneous IL-1, secreted in response to disease or stress, may alter the endogenous opioid pathway by inducing the expression of opioid receptors in endothelial cells. Clinically, the potential impact on the body's defense mechanisms resulting from the interaction between opioids, such as morphine, and cytokines, such as IL-1, can be substantial. Further investigation of this interaction is essential to understanding the extent of damage in human body caused by drugs of abuse, such as morphine.

Animals↗
PubMed1998

Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea.

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/beta2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/beta2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1beta reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.

Animals↗
PubMed1997

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. II. Evidence for persistent chronic rejection despite high levels of donor microchimerism.

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.

Animals↗
PubMed1997

Oct-1 and CCAAT/enhancer-binding protein (C/EBP) bind to overlapping elements within the interleukin-8 promoter. The role of Oct-1 as a transcriptional repressor.

Interleukin-8 (IL-8), a potent neutrophil chemoattractant, can be expressed at high levels by many different cell types after immune stimulation. In contrast, expression of IL-8 in these same cells is virtually absent in the unstimulated state, demonstrating the tight regulation of the IL-8 gene. Although much is known about how this gene is transcriptionally activated after immune stimulation, little is known about the regulation of the IL-8 promoter in the absence of immune activation. In this study we examine how the IL-8 promoter is transcriptionally regulated in the uninduced state and how these mechanisms are altered in response to immune stimulation by IL-1beta. Electrophoretic mobility shift assay and transfection studies show that the IL-8 promoter is transcriptionally regulated by both positive and negative elements. Although the nuclear factor-kappaB (NFkappaB) element regulates only inducible activity of the IL-8 promoter in response to stimulation with IL-1beta, the AP-1 and CCAAT/Enhancer-binding Protein (C/EBP) elements influence both basal and inducible activities. In contrast to these three positive regulatory elements, the binding of the ubiquitously expressed POU-homeodomain transcription factor, Oct-1, strongly represses transcriptional activity of the IL-8 promoter by binding independently to an element overlapping that of C/EBP.

Base Sequence↗
PubMed1997

Inhibition of IL-8 gene expression in Caco-2 cells by compounds which induce histone hyperacetylation.

Ulcerative colitis, an idiopathic inflammatory disease of the colonic mucosa, can be effectively treated by enemas containing short chain fatty acids (SCFA) such as butyrate, propionate, and acetate. The molecular mechanisms that lead to this response have not been well characterized. It is well known that intestinal inflammation leads to an alteration in patterns of epithelial differentiation with an increase in epithelial proliferation and an expansion of cell populations in an undifferentiated state. SCFAs such as butyrate are capable of inhibiting cell proliferation and inducing a differentiated phenotype in vitro. The Caco-2 colon cancer cell line was used to study the effect of SCFAs and the process of cellular differentiation on the expression of the pro-inflammatory cytokine, interleukin 8 (IL-8). SCFAs and trichostatin A, structurally unrelated compounds which both induce histone hyperacetylation, both led to a dose-dependent inhibition of IL-8 gene expression. Furthermore, spontaneous differentiation of Caco-2 cells by growth to a post-confluent state also inhibited the expression of IL-8. A possible mechanism by which SCFAs may be effective in the treatment of ulcerative colitis may be through their ability to increase histone acetylation states and inhibit the production of pro-inflammatory substances by the intestinal epithelium.

Acetylation↗
PubMed1996

[Genetic control of humoral immune response to xenografts. A monoclonal antibody that causes the hyperacute rejection of cardiac xenografts uses the genes in a native form].

The early phases of the rejection of xenografts exchanged between closely-related species are dominated by a vigorous humoral immune response. We have recently used a linkermediated polymerase chain reaction to generate Ig heavy and light chain specific cDNA libraries to examine the Ig gene control of a prototypic IgM monoclonal antibody, HAR-1, that causes the hyperacute rejection of hamster xenografts. Recombinant clones from the library were screened directly from bacterial colonies by PCR and the nucleic acid sequences of the clones established. Our results demonstrate that the HAR-1 hybridoma is encoded by Ig VH and JH genes in a germline configuration. Comparison of the cDNA sequence for HAR1-VH with the germline equivalent of the gene isolated from newborn LEW liver (provisionally designated VH1.1) showed that the two VH sequences share a nucleic acid identity of 99,3%. Similarly, the HAR-1 monoclonal uses a Ig JH gene that is 98,2% identical with the JH1 nucleic acid sequence available in the GenBank. The use of Ig VH and JH genes in a germline configuration is similar to that seen with polyreactive natural antibodies to infectious agents and autoantibodies. These humoral responses are thought to be the results of the stimulation of a T cell-independent subset of B cells, the B-1a/B-1b subset, that are responsible for producing antibodies that serve as a primitive humoral (natural antibody) defense mechanism against infectious diseases. Our results suggest that the humoral component of the rejection of xenografts in the hamster-to-rat model may represent the stimulation of this type of B cell antibody response by xenogeneic target antigens that share antigenic epitopes with bacteria and other infectious agents.

Animals↗
PubMed1995

Genetic control of the humoral immune response to xenografts. I. Functional characterization of rat monoclonal antibodies to hamster heart xenografts.

The rejection of cardiac xenografts in the hamster-to-rat combination is characterized by the production of IgM antibodies that result in the rapid loss of the graft. We have recently produced rat monoclonal antibodies (mAb) to hamster heart xenografts in an attempt to develop reagents for use in identifying the target antigens for this reaction and to study the nature of the genetic control of the humoral response. The monoclonals were created by the fusion of myeloma cells with splenic lymphocytes from LEW rat recipients of hamster cardiac xenografts. The hybridomas were screened for antibody production, reactivity to hamster cell surface antigens, and the ability to mediate hyperacute rejection of hamster heart xenografts. A panel of monoclonal antibodies has been identified that are capable of inducing hyperacute rejection. All of these mAbs are IgM and bind strongly to hamster vascular endothelium. None of the mAbs were lymphocytotoxic or bound to hamster lymphocytes or erythrocytes. Immunopathologic studies demonstrated that these mAbs react specifically with hamster vascular endothelium and mediate a complement-dependent humoral reaction leading to the destruction of the cardiac xenografts. One of the mAbs (designated as HAR-1) has been characterized in detail. HAR-1 detects antigens distributed in the vascular endothelium, epithelium of bronchi in the lung, small intestine, tubules of kidney, and selective components of lymphoid organs--e.g., the stromal cells of the spleen and thymic medullary epithelium. Western blot analysis of hamster heart proteins with HAR-1 showed multiple bands with two major bands migrating at 80 kDa and 48 kDa. Absorption of the HAR-1 antibody with 48 individual carbohydrate molecules demonstrated that the strongest reactivity of the antibody is with a sialyl-Lea carbohydrate antigen.

Animals↗
PubMed1995

Genetic control of the humoral immune response to xenografts. II. Monoclonal antibodies that cause rejection of heart xenografts are encoded by germline immunoglobulin genes.

The early phases of the rejection of xenografts exchanged between closely related species are dominated by a vigorous humoral immune response. We have recently used a linker-mediated polymerase chain reaction (LM-PCR) to generate Ig heavy and light chain-specific cDNA libraries to examine the Ig gene control of a prototypic IgM monoclonal antibody, HAR-1, that causes the hyperacute rejection of hamster xenografts. Recombinant clones from the library were screened directly from bacterial colonies by PCR and the nucleic acid sequences of the clones established. Our results demonstrate that the HAR-1 hybridoma is encoded by Ig VH and JH genes in a germline configuration. Comparison of the cDNA sequence for HAR-1 VH with the germline equivalent of this gene isolated from newborn LEW liver (provisionally designated VHHAR-1) showed that the two VH sequences share a nucleic acid identity of 99.3%. Similarly, the HAR-1 monoclonal uses a Ig JH gene that is 98.2% identical with the JH1 nucleic acid sequence available in the GeneBank. The use of Ig VH and JH genes in a germline configuration is similar to that seen with polyreactive natural antibodies to infectious agents and autoantibodies. These humoral responses are thought to be the result of the stimulation of a T cell-independent subset of B cells, the B-1a/B-1b subset, that is responsible for producing antibodies that serve as a primitive humoral (natural antibody) defense mechanism against infectious diseases. Our results suggest that the humoral component of the rejection of xenografts in the hamster-to-rat model may represent the stimulation of this type of B cell antibody response by xenogeneic target antigens that share antigenic epitopes with bacteria and other infectious agents.

Amino Acid Sequence↗
PubMed1995

The use of a pig liver xenograft for temporary support of a patient with fulminant hepatic failure.

A 26-year-old female patient with fulminant hepatic failure and a history of autoimmune hepatitis was heterotopically transplanted with a pig hepatic xenograft to provide temporary metabolic support prior to transplantation with a human donor organ. Circulating natural antipig antibodies were removed prior to transplantation by plasmapheresis and ex vivo en bloc perfusion of the donor pig kidneys. The liver xenograft functioned after transplantation as measured by active bile production, stabilization of prothrombin levels, and reduction in the circulating levels of lactic acid and the enzymes AST and ALT. Despite the removal of greater than 90% of the recipient's natural xenoantibodies prior to transplantation, the levels of antibody rapidly returned and were associated with antibody and complement-mediated rejection of the donor graft. Immunohistochemical evidence of graft rejection could be detected by the deposition of antibody, complement components including properdin, and endothelial swelling as early as 3 hr posttransplantation. These lesions progressed in severity and were accompanied by evidence of thrombosis and ischemic necrosis of the liver xenograft by 34 hrs posttransplantation. The main portal vein, hepatic artery, and vena cava were patent. The placement of the liver graft did not result in any improvement in the neurological status of the patient and she died 34 hr after xenografting due to irreversible brain damage. The information derived from this case has renewed interest in the clinical use of bioartificial devices and whole organ perfusion using xenogeneic tissue for temporary bridging of patients prior to allografting.

Adult↗