Recent advances in experimental microsurgery: the 4th congress of the international society for experimental microsurgery (ISEM)
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Journal Article↗
Biomedical subjects
R Zhong
Publications and source records attributed to R Zhong.
Effect of major histocompatibility complex expression on murine intestinal graft survival.
BACKGROUND: Clinical intestinal transplantation has been plagued by frequent and severe graft rejection. It has been proposed that the major histocompatibility complex (MHC) antigens might play a critical role in this process owing to their extensive expression on enterocytes and mucosa-associated immune cells. METHODS: The present study examined the role of MHC antigens in intestinal graft rejection using MHC class I-deficient and MHC class II-deficient donors. RESULTS: Grafts with normal MHC expression were rejected by 9 days, whereas survival was prolonged to 14 days in the MHC class II-deficient grafts (P=NS) and to 20 days in the MHC I-deficient grafts (P<0.002). In all groups, early rejection was characterized by (1) increased crypt cell apoptosis, as detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique of in situ labeling; and (2) the increased expression of perforin and a CD8 phenotype in the graft-infiltrating cells. CONCLUSIONS: These data suggest that MHC antigens, CD8-positive T cells, and perforin-expressing cells contribute to intestinal graft rejection. Apoptosis of the progenitor epithelial crypt cells during early intestinal rejection may impair the gut's ability to regenerate and repair mucosal damage.
Animals↗
Treatment of cardiac allografts with established leukocyte infiltration by modulation of alpha4 (CD49d) and leukocyte function-associated antigen-1 (CD11a/CD18) integrin function.
BACKGROUND: Leukocyte infiltration is a landmark feature of organ rejection. The present study was undertaken to determine whether monoclonal antibodies (mAb) against alpha4 (CD49d) and/or leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) would reverse ongoing rejection in a mouse C57BL/6-to-BALB/c heart transplant model. METHODS: Control animals had rejection on postoperative day (POD) 8. Treatment with mAb started on POD 4 when leukocyte infiltration was well established. The recipients were treated with (1) mAb LFA-1, (2) mAb alpha4, and (3) mAbs LFA-1 + alpha4 at a dose of 6 mg/kg/day i.v. on PODs 4, 5, and 7. Untreated and rat IgG-treated animals were used as controls. RESULTS: Control animals experienced rejection on POD 8. Treatment with mAb against LFA-1 or alpha4 alone prolonged allograft survival to 17.0+/-3.2 and 24.3+/-4.6 days, respectively (P < 0.01 vs. controls). Combination therapy with both mAb increased allograft survival to 28.2+/-3.7 days (P < 0.01 vs. controls). Sequential pathological studies showed the mAb to alpha4, but not LFA-1, markedly reduced the degree of lymphocytic infiltration in cardiac allografts. In contrast, a different pattern was observed using in vitro studies: mAb to LFA-1, not alpha4, significantly reduced proliferative responses in mixed lymphocyte culture and interleukin-2 production from recipient splenocytes on POD 8. CONCLUSION: These data indicate that integrins play an important role in rejection. Although the effect of mAb against alpha4 and LFA-1 may involve different mechanisms, treatment with mAbs to integrins may be valuable in future clinical transplantation by averting ongoing rejection and prolonging graft survival.
Animals↗
Transcriptional activity of heat shock factor 1 at 37 degrees C is repressed through phosphorylation on two distinct serine residues by glycogen synthase kinase 3 and protein kinases Calpha and Czeta.
Heat shock factor 1 (HSF1) is the key transcriptional regulator of the heat shock genes that protect cells from environmental stress. However, because heat shock gene expression is deleterious to growth and development, we have examined mechanisms for HSF1 repression at growth temperatures, focusing on the role of phosphorylation. Mitogen-activated protein kinases (MAPKs) of the ERK family phosphorylate HSF1 and represses transcriptional function. The mechanism of repression involves initial phosphorylation by MAP kinase on serine 307, which primes HSF1 for secondary phosphorylation by glycogen synthase kinase 3 on a key residue in repression (serine 303). In vivo expression of glycogen synthase kinase 3 alpha or beta thus represses HSF1 through phosphorylation of serine 303. HSF1 is also phosphorylated by MAPK in vitro on a second residue (serine 363) adjacent to activation domain 1, and this residue is additionally phosphorylated by protein kinase C. In vivo, HSF1 is repressed through phosphorylation of this residue by protein kinase Calpha or -zeta but not MAPK. Regulation at 37 degrees C, therefore, involves the action of three protein kinase cascades that repress HSF1 through phosphorylation of serine residues 303, 307, and 363 and may promote growth by suppressing the heat shock response.
3T3 Cells↗
Surgical technique for vascularized ear transplantation in mice.
Traditionally, the mouse nonvascularized skin graft has been widely used in organ transplant research. There are, however, several limitations with this model, the main one being the different immune response of vascularized vs. nonvascularized grafts. We have recently developed a vascularized, orthotopic ear transplant model in mice. This model has several advantages that make it a useful tool to study many aspects of transplantation. The donor operation consists of harvesting the ear with intact arterial and venous pedicles. The arterial pedicle is dissected down to the common carotid artery with ligation of all branches except the auricular artery. The venous pedicle is based on the jugular vein with preservation of the auricular veins draining into the posterior facial vein. In the subsequent recipient operation, the graft is transplanted into an orthotopic position using the common carotid artery for arterial inflow and the jugular vein for venous outflow. We performed 16 such transplants (6 isografts, 5 allografts, and 5 xenografts), with a success rate of 94% (15/16). The donor operation time was 1.5 hours and the recipient operation time was 2.5 hours. The total time for anastamosis was 45 +/- 5 minutes. Serial biopsies were obtained on days 3, 5, and 7, and correlated with gross findings. In summary, this study illustrates that it is technically possible to transplant a mouse ear graft. The main advantage of this model is that it is a vascularized graft that can be visibly observed and easily biopsied, thus allowing for good correlation between gross and histological findings after transplantation. We plan to use this model further to study in detail the rejection patterns in an allograft and xenograft setting.
Animals↗
Strategies to prevent thrombosis in xenotransplants.
Coagulation remains a major barrier to successful xenotransplantation. Identification of the role of complement activation in HAR and prevention of complement activation by high expression of DAF and CD59 has largely overcome HAR. Understanding the molecular basis of DXR and identification of novel strategies to prevent activation of endothelial cells using monoclonal antibodies, soluble inhibitors of coagulation, vaccination, antisense constructs, and recombinant DNA technology offers the promise of overcoming the thrombosis associated with xenotransplantation.
Acute Disease↗
Rat-to-mouse small bowel xenotransplantation: novel models to study hyperacute and acute humoral rejection.
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Acute Disease↗
Beneficial effects of microsurgical lymphatic reconstruction after intestinal transplantation in rats.
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Animals↗
Glucose turnover and insulin sensitivity in rats with pancreatic islet transplants.
To study the metabolic effects of insulin derived from islet grafts, oral glucose tolerance (OGT) and glucose turnover were examined in streptozotocin-induced diabetic Lewis rats rendered normoglycemic by syngeneic islet grafts in the renal subcapsular space (REN), in REN with renal vein-to-mesenteric vein anastomosis (REN-RMA), in the liver (intrahepatic [IH]), or in a parahepatic omental pouch (POP) and compared with normal rats. Normal OGT was found at 1 month posttransplant in all animals receiving approximately 3,000 islets, with hyperinsulinemic responses in the REN group compared with the other groups, and with higher C-peptide responses in the IH group than in the other groups (P < 0.05 by one-way analysis of variance). Glucose turnover studies in the insulin-stimulated steady state (INS-SS; infusion of insulin at 10 pmol x kg(-1) x min(-1)) at 2 months posttransplant showed that whole body glucose disappearance rates (Rd) were similar in all groups, but the REN group had higher steady-state insulin levels than the other groups. Glucose infusion rates (GIRs) were lower in the REN and IH groups than in the other groups. Apparent endogenous glucose production (EGP) was not completely inhibited in the REN and IH groups, while complete inhibition was observed in the other groups. When INS-SS insulin levels were matched to the level in REN rats by increasing the insulin infusion rate to 20 pmol x kg(-1) x min(-1) in REN-RMA, IH, and normal rats, GIR and Rd were elevated, exceeding those values in REN rats, but GIR in IH rats was still lower than in REN-RMA and normal rats. Thus, 1) in the REN group, impairment of inhibition of EGP and of stimulation of Rd by exogenous insulin contribute to insulin resistance; 2) in the IH group, incomplete inhibition of EGP is the major determinant of insulin resistance; and 3) with portal delivery of insulin in the REN-RMA and POP groups, normal insulin sensitivity is preserved. The present study confirms that hepatic portal delivery of islet secretions is necessary for physiological regulation of glucose metabolism. The study also suggests the IH grafts do not provide physiological regulation of glucose metabolism, raising the question of whether the liver is an appropriate site for insulin-secreting tissue replacement therapy in diabetes.
Anastomosis, Surgical↗
[Overexpression of cyclin D1 in laryngeal carcinomas].
Cyclin D1 is a set of periodic protein which governs G1 progression. Cyclin D1 gene is localized on chromosome 11q13 which encodes a 295-aa protein. Overexpression of cyclin D1 leads to abnormal cellular proliferation. Which underlies processes of tumorigenesis. In this paper twenty-five fresh specimens of laryngeal carcinomas were examined by means of immune flurescence technique. Overexpression of cyclin D1 was found in 9 of 16 laryngeal carcinomas (37%). There was no statistical correlation between overexpression of cyclin D1 and TNM staging, differentiation grading (P > 0.05). Normal tissue adjacent to tumors lacked any detectable cyclin D1 expression or rare scattered positive cells. It was likely that cyclin D1 overexpression wasn't an early event in processes of tumorigenesis and tumor progression. Follow-up investigation demonstrated that tumors recurred in 4 of 9 primary tumors overexpressing cyclin D1. But only 1 of 16 that expressed cyclin D1-negative. There was statistical significance between them, so overexpression of cyclin D1 could serve as a new prognostic marker.
Cyclin D1↗
CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells.
Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase-CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.
Amino Acid Sequence↗
Human breast cancer growth inhibited in vivo by a dominant negative pleiotrophin mutant.
Pleiotrophin (PTN) is a recently described 18- kDa heparin binding growth/differentiation factor. It also is a proto-oncogene; cells transformed by the Ptn gene form highly angiogenic tumors when implanted into the nude mouse. PTN may be an important regulator of transformation in other tumors, because constitutively high levels of expression of the pleiotrophin (Ptn) gene are found in human breast cancer and other malignant cell lines, and its levels of expression are high in many human tumor specimens. To determine whether PTN is an important regulator of the malignant phenotype of human breast cancer cells, we constructed a mutant cDNA to encode a truncated PTN designed to heterodimerize with the product of the endogenous Ptn gene during processing. The mutant gene product blocked transformation of NIH 3T3 cells by the wild type (wt) Ptn gene product. The mutant Ptn cDNA was then introduced into human breast cancer MDA-MB-231 cells, and clonal lines that stably express the mutant Ptn cDNA were selected. The truncated PTN was shown to form heterodimers with the endogenous Ptn gene product in these cells. Furthermore, the MDA-MB-231 cells that express the mutant Ptn gene were no longer transformed; they failed to form plaques or colonies in soft agar and were unable to form tumors in the athymic nude mouse. The results establish an important role of PTN in the dysregulated growth of human breast cancer cells and suggest that constitutive expression of PTN may be essential to the malignant phenotype of human breast cancers in vivo.
3T3 Cells↗
Budesonide, a locally acting steroid, prevents graft rejection in a rat model of intestinal transplantation.
BACKGROUND: The requirement for potent systemic immunosuppression to prevent intestinal graft rejection has resulted in high rates of infection and post-transplant lymphoproliferative disease. Budesonide (BUD) is a locally acting steroid that is almost completely metabolized during its first pass through the intestinal wall and liver. The present study examined whether BUD could prevent small bowel allograft rejection without causing the adverse systemic effects associated with prednisolone. METHODS: Orthotopic Brown Norway to Lewis rat small bowel allotransplants were randomly assigned to treatment with low-dose BUD (0.1 mg/kg/day, p.o.) and high-dose BUD (1.0 mg/kg/day, p.o.) with and without cyclosporine (CsA; 2 mg/kg/day s.c.). The following parameters were assessed: allograft survival, recipient plasma adrenocorticotrophic hormone (ACTH) levels, recipient adrenal, splenic and thymic weights, recipient CsA levels, and graft histopathology. RESULTS: Low- and high-dose BUD alone did not prolong graft survival compared with the untreated group (9.1+/-0.4 days vs. 11+/-0.8 days vs. 9.7+/-0.4 days, respectively). However, when low-dose BUD and high-dose BUD were given in combination with CsA, the mean graft survival times were prolonged to 27.6+/-5.3 and 36.6+/-8.0 days, respectively (P<0.01). ACTH levels, adrenal weights, and thymic weights were not significantly different in the treatment and control groups receiving intestinal transplants. CONCLUSIONS: BUD enhances the immunosuppressive effects of CsA and prolongs small bowel allograft survival in rats without inhibiting normal ACTH release. These data suggest that BUD may be a useful immunosuppressive agent for clinical intestinal transplantation.
Adrenocorticotropic Hormone↗
Assessment of the longevity of the liver using a rat transplant model.
To assess the longevity of the liver, arterialized, orthotopic liver grafts were performed using syngeneic male BN/BiRij rats. Young (5-month-old) livers were transplanted into 5-month-old recipients (group I, n = 27), and old (28-month-old) livers were transplanted into 5-month-old rats (group II, n = 28). Recipient survival after transplantation was similar in both groups. The average age of the livers at the time of death was 16.7 months in group I and 39.1 months in group II. Four of the livers in group II survived for more than 4 years (48.1 to 52.4 months). Early deaths (less than 1 year) after transplantation were most commonly caused by biliary obstruction and cholangitis in both groups. Late deaths (more than 1 year) after grafting were mainly from heart failure or tumors. None of the animals died of liver failure or liver disease. Weight gain in the rats, total serum protein levels, and alanine transaminase levels after transplantation did not differ significantly between the two groups. There was a trend for the histological features of aging of the liver-fibrosis, bile duct proliferation, and pigment deposition-to become more prevalent as the livers became very old (mean age, 46 months). Nevertheless, typical aging changes, as individual findings, were absent in nearly half of the oldest organs. The alterations in morphology had no apparent effect on the ability of the livers to sustain the lives of the recipients. The liver of the BN/BiRij rat was capable of surviving far beyond the maximum life span of BN/BiRij rats, and rats in general. It did not become diseased in the process.
Age Factors↗
Generation of dendritic cell-like antigen-presenting cells in long-term mixed leucocyte culture: phenotypic and functional studies.
The mechanisms contributing to the proliferation and differentiation of antigen-presenting cell (APC) precursors upon antigen stimulation or tissue injury are poorly understood. Herein, we report the induction of a population of dendritic cell-like cells (DLC) with potent antigen-presentation function from unfractionated spleen cells by means of repetitive allostimulation in long-term mixed leucocyte cultures (LT-MLC). Initially, only a few adherent DLC were observed. By 4-6 weeks, however, there were large numbers of DLC which survived persistently. Features of these DLC are closely related to dendritic cells (DC), including (1) dendritic, veiled or spiny-processed morphology; (2) expression of a wide array of leucocyte surface markers including DC-associated or restricted antigens: 33D1, NLDC-145, CD11c (N418), heat-stable antigen (HSA), CD44, B7-1 and B7-2; (3) ability to migrate to draining lymph nodes and white pulp area of spleen; (4) expression of high level of major histocompatability complex (MHC) class II molecules and (5) more potent mixed leucocyte reaction (MLR)-stimulating capacity than peritoneal macrophages and APC-enriched spleen cells. DLC-stimulated MLR was inhibited by monoclonal antibodies (mAbs) to B7-1, B7-2, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), leucocyte-function associated antigen-1 (LFA-1) or very-late activation antigen-4 (VLA-4) by 30-55%. When maintained for more than 2 months, the DLC did not lose their MLR-stimulating activity, but many surface markers were down-regulated except for Mac-2 and VCAM-1, which remained stable or were up-regulated, respectively. In short-term culture, the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-2 enhanced proliferation of DLC, while tumour necrosis factor-alpha (TNF-alpha) and IL-4 did not. IL-4 suppressed not only 'spontaneous', but also GM-CSF-enhanced proliferation, suggesting that cytokines play a differential role in DLC proliferation. These results confirm that professional APC can proliferate in response to repetitive antigen stimulation, and their proliferation is differentially regulated by cytokines. A comparison study of DLC with typical DC is being carried out in our laboratory.
Animals↗
Molecular cloning and gene expression analysis of PSP94 (prostate secretory protein of 94 amino acids) in primates.
Prostate secretory protein of 94 amino acids (PSP94) has shown the potential to be a diagnostic biomarker and a therapeutic agent for prostate cancer. Primates have been the main animal models for studying the biology of this molecule. We have cloned and analyzed the cDNA and promoter region of PSP94 from baboon (Papio anubis). Sequence divergence among baboon, monkey, pig, and human, in both the exons and 5'-flanking region indicates rapid evolution of the PSP94 gene. There are conserved steroid hormone response elements (SHRE) in the promoter region of all three primate species. Multiple, alternative transcripts starting near these SHREs and upstream to the TATA box were identified by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of 5'-cDNA ends (5' RACE) in primate prostatic tissues. This differential transcription initiation may be linked to androgen regulation of PSP94 gene expression. PSP94 transcripts were detected by RT-PCR in a wide variety of mucus-secreting tissues. However, the alternative transcripts were found only in the prostate. The distribution of the PSP94 protein in baboon secretory tissues was also examined by Western blot analysis using a polyclonal antibody against the human homolog. A positive immunoreactive band was detected, but it was weak, due probably to epitope divergence between the two species. In all young, healthy primate animals tested, the level of immunoreactive PSP94 in prostate tissues was lower than expected. In addition, RT-PCR combined with Southern blot analysis on prostate tissues in these animals failed to detect the PSP57 mRNA produced by alternative splicing of PSP94 primary transcript. These observations can be explained by the sexual immaturity and incomplete prostate development in these young primates. This explanation was supported by histological examination of their prostate during PSP94 immunohistochemistry.
Amino Acid Sequence↗
Disruption of interfascicular fiber differentiation in an Arabidopsis mutant.
Arabidopsis develops interfascicular fibers in stems for needed support of shoots. To study the molecular mechanisms controlling fiber differentiation, we isolated an interfascicular fiber mutant (ifl1) by screening ethyl methanesulfonate-mutagenized Arabidopsis populations. This mutant lacks normal interfascicular fibers in stems. Interestingly, some interfascicular cells were sclerified in the upper parts but not in the basal parts of the ifl1 stems. These sclerified cells were differentiated at a position different from that of interfascicular fibers in the wild type. Lack of interfascicular fibers correlated with a dramatic change of stem strength. Stems of the mutant could not stand erect and were easily broken by bending. Quantitative measurement showed that it took approximately six times less force to break basal stems of the mutant than of the wild type. In addition, noticeable morphological changes were associated with the mutant, including long stems, dark green leaves with delayed senescence, and reduced numbers of cauline leaves and branches. Genetic analysis showed that the ifl1 mutation was monogenic and recessive. The ifl1 locus was mapped to a region between the 17C2 and 7H9L markers on chromosome 5. Isolation of the ifl1 mutant provides a novel means to study the genetic control of fiber differentiation.
Arabidopsis↗
Insulin resistance prevented by portal delivery of insulin in rats with renal subcapsular islet grafts.
We determined the metabolic effects of insulin derived from renal subcapsular islet grafts, either with systemic delivery of insulin through renal venous drainage (REN) or with portal delivery of insulin after renal vein-to-superior mesenteric vein anastomosis (RMA), in streptozotocin-induced diabetic Lewis rats, in comparison with normal rats. After gavage glucose, the plasma glucose responses were similar to normal in REN and RMA rats; however, hyperinsulinemia occurred in REN rats (area under the concentration curves [AUCs] of insulin, 27 +/- 3 nmol x 1(-l) min) in comparison with RMA (14 +/- 2) and normal rats (19 +/- 2), P < 0.003, with no difference in C-peptide responses. The ratio of AUC C-peptide to AUC insulin was lower in REN (2.0 +/- 0.2) than in RMA (3.4 +/- 0.3) and normal animals (3.2 +/- 0.3), P < 0.0005. In euglycemic-hyperinsulinemic clamp studies using the same insulin infusion rate (10 pmol x kg(-1) x min(-1), insulin resistance was found in REN animals (mean glucose infusion rate [GIR], REN: 7.5 +/- 1.2; RMA: 12.0 +/- 1.2; normal: 12.7 +/- 1.0 mg x kg(-1) x min(-1); P < 0.008), with higher steady-state insulin levels in REN (554 +/- 63 pmol/l) than in RMA (291 +/- 26) and normal rats (269 +/- 60), P < 0.0001. With matching steady-state insulin levels in RMA and REN rats during infusion of insulin at 20 pmol x kg(-1) x min(-1) in RMA rats (steady-state insulin 623 +/- 64 pmol/l), GIR was 15.7 +/- 0.7 mg x kg(-1) x min(-1). Thus, systemic delivery of insulin from islet grafts is associated with hyperinsulinemia, insulin resistance, and decreased metabolic clearance of insulin. These abnormalities are prevented by portal delivery of insulin from islet grafts in the same site. The findings are consistent with the hypothesis that portal delivery of insulin is important in maintenance of normal whole-body insulin sensitivity.
Anastomosis, Surgical↗