Laparoscopic kidney orthotopic transplant: preclinical study in the pig model.

BACKGROUND: Laparoscopic surgery has rapidly expanded in clinical practice replacing conventional open surgery over the last three decades. Laparoscopic donor nephrectomy has been favored due to its multiple benefits. The aim of this study was to explore the safety and feasibility of kidney transplantation by a laparoscopic technique in a pig model. MATERIALS AND METHODS: The study was approved by the university animal ethics committee. Eight female pigs (Sus Scrofra, weighing 45-50 kg) were divided into 2 groups: group I included 4 animals that underwent laparoscopic kidney orthotopic transplantation on the left side. The right kidney was remained functional in situ. The pigs recovered and were observed for 1 week. In the 4 hosts group II pigs underwent a laparoscopic kidney transplantation on the left side. With simultaneous clipping of the right ureter. After recovery, the pigs were observed for 4 weeks. A laparotomy for examination was performed prior to euthanasia. RESULTS: All 4 group I pigs survived for 1 week. The laparotomy showed normal graft perfusion with wall patent renal artery and vein as well as satisfactory urine output upon transection of ureter in 3 hosts. Renal artery stenosis occurred in one pig. In The Immediate kidney graft function was achieved in 3 group II pigs. The fourth died following extubation due to laryngospasm despite a functional graft. The average creatinine levels were 195.5 µmol/L on day 3; 224.5 µmol/L at week 1; 127 µmol/L at week 2; 182.7 umol/L at week 3; and 154.7 umol/L at week 4. CONCLUSION: Laparoscopic kidney transplantation was feasible and safe in a pig model with immediate graft function. This study will provide further evidence to support application of laparoscopic technique to human kidney transplant.

Levels of Ly-49 receptor expression are determined by the frequency of interactions with MHC ligands: evidence against receptor calibration to a "useful" level.

Ly-49 receptor expression was studied in NK cells that developed in fully MHC-mismatched mixed bone marrow chimeras, in which host and donor MHC ligands were expressed solely on various proportions of hemopoietic cells or on both hemopoietic and nonhemopoietic cells. When hemopoietic cells were the only source of MHC ligand, a strong correlation between the level of down-regulation of Ly-49A, Ly-49C, and Ly-49G2 and the number of hemopoietic cells expressing their MHC ligands was observed on both donor and host NK cells. In some animals with low levels of donor hemopoietic chimerism, NK cells of donor origin expressed Ly-49 receptors at higher levels than was observed in normal mice of the same strain. This unexpected observation is inconsistent with the receptor calibration theory, which states that expression of Ly-49 inhibitory receptors is calibrated to an optimal level to maintain an NK cell repertoire that is sensitive to perturbations in normal class I ligand expression. Our data suggest a model in which Ly-49 receptors down-modulate in accordance with the frequency of their interactions with ligand-bearing cells, rather than a model in which these receptors calibrate to a specific "useful" level in response to ligands present in their environment.

Altered expression of Ly-49 receptors on NK cells developing in mixed allogeneic bone marrow chimeras.

Ly-49 molecules are used by NK cells to distinguish 'self' from 'non-self', but the determinants of Ly-49 expression that allow this distinction to be made are not understood. The education of NK cells for self/non-self recognition was studied in murine mixed allogeneic bone marrow chimeras, in which NK cells are of both host and donor origin. Marked alterations in Ly-49 receptor expression were observed on both host and donor NK cells developing in BALB/c --> B6 mixed chimeras. Ly-49A and Ly-49G2 expression was lower on host B6 NK cells of mixed chimeras compared to non-transplanted B6 controls. Among donor BALB/c NK cells, Ly-49C expression levels were reduced, but the proportion of Ly-49C+ cells was increased, whereas Ly-49G2 expression was up-regulated compared to non-transplanted BALB/c controls. Thus, Ly-49 expression on donor and host NK cells developing post-bone marrow transplantation evolves toward the expression pattern of the host and donor strains respectively, due to the presence of the allogeneic MHC. In vitro functional NK cell assays showed that donor NK cells in mixed chimeras were not tolerant to host antigens and that host NK cells were not tolerant to the donor. Our data are consistent with a model in which MHC expression in the environment has a dominant down-regulating effect on the expression of Ly-49 molecules that recognize those MHC molecules, regardless of whether they are self or allogeneic. This down-regulation, combined with the limited repertoire of Ly-49 molecules, may not be sufficient to allow NK cells to be tolerant of MHC antigens of a fully MHC-mismatched allogenic strain.

Glycosylphosphatidylinositol-anchored H-2Db molecules are defective in antigen processing and presentation to cytotoxic T lymphocytes.

Glycosylphosphatidylinositol-anchored (GPI)-Db molecules are defective in mediating cytotoxic T lymphocytes (CTL) lysis of transfected lymphoma cells, compared to their transmembrane (TM) counterpart. This defect is manifest when antigenic peptide must be processed and presented through the endogenous pathway. These same transfectants can be lysed by allospecific CTL, or by antigen-specific Db-restricted CTL when pulsed with appropriate exogenous synthetic peptide, demonstrating that they can bind and present peptide for CTL-mediated lympholysis. The defect apparently results from differences between GPI-Db and TM-Db assembly and transport, or from differences in membrane topology that affect CD8+ CTL recognition of major histocompatibility complex/peptide complex.

MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide-regulated interaction with TAP via their extracellular domains.

Newly assembled heavy chain-beta 2m heterodimers of class I histocompatibility molecules associate with the endoplasmic reticulum (ER) peptide transporter, TAP, and subsequently dissociate from TAP in parallel with their transport from the ER to the Golgi apparatus. It appears that TAP-associated class I molecules are waiting to bind appropriate peptides before they dissociate from TAP and leave the ER since binding of high affinity peptides to class I molecules in vitro leads to dissociation of TAP-class I complexes. In further support of this notion, we report that limiting peptide supply through inhibition of proteasome activities prolongs the association of mouse class I molecules with TAP and concomitantly slows their transport to the Golgi apparatus. By using a series of deletion mutants and hybrid class I molecules we demonstrate that the extracellular domains of class I molecules are sufficient for their peptide-regulated interaction with TAP. Furthermore, based on the inability of an alpha 3 domain-specific mAb to recognize TAP-class I complexes and the fact that a point mutant of the Dd molecule at residue 222 is unable to bind to TAP, it is likely that a major site of interaction with TAP resides in the membrane-proximal region of the heavy chain alpha 3 domain. Finally, we examined the relationship between the interaction of mouse heavy chain-beta 2m heterodimers with TAP and with the resident ER chaperone, calnexin. Most heterodimers that bound to TAP were found to associate simultaneously with calnexin. Upon delivery of peptide to class I molecules in permeabilized cells, dissociation from TAP was observed but the interaction with calnexin was largely maintained. Therefore, both TAP and calnexin may participate in the ER retention of peptide-deficient class I molecules. However, since release from calnexin occurs after dissociation from TAP, it appears that calnexin ultimately determines if a class I molecule is to be exported from the ER.

Sequence and transcription of Qa-2-encoding genes in mouse lymphocytes and blastocysts.

The protein product of the mouse preimplantation embryo development (Ped) gene, which controls the rate of preimplantation embryonic cleavage division and subsequent embryo survival, is the Qa-2 antigen. This major histocompatibility complex (MHC) class I b protein is encoded by four genes, Q6, Q7, Q8, and Q9. The present study was undertaken to begin to elucidate which of the four Qa-2-encoding genes are responsible for the Ped gene phenotype in the C57BL/6 mouse (H2(b)). First, restriction maps of the four genes, using 25 restriction enzymes, were created. The RE maps confirmed that Q6 is similar to Q8 and Q7 is similar to Q9, but that the Q6/Q8 gene pair differs from the Q7/Q9 gene pair. The genomic DNA sequences of Q6 and Q8 were determined, as well as the DNA sequences of exons 4 - 8 of Q9, and the 5' regulatory regions of Q6, Q8, and Q9. This DNA sequence information, combined with the published DNA sequence information for the entire Q7 gene and exons 1 - 3 of Q9, allowed us to design primers for reverse transcription-polymerase chain reaction that could distinguish which of the four genes were transcribed in mouse lymphocytes and embryos. It was found that all four genes are transcribed in lymphocytes, but only Q7 and Q9 are transcribed in mouse embryos. Thus, both Q7 and Q9 are candidates for the genes responsible for the Ped gene phenotype.

Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo.

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.

Correction of the class H defect in glycosylphosphatidylinositol anchor biosynthesis in Ltk- cells by a human cDNA clone.

Previous attempts to express glycosylphosphatidylinositol-anchored proteins in Ltk- cells have not been successful because Ltk- cells cannot synthesize N-acetylglucosamine-phosphatidylinositol, the first intermediate in anchor biosynthesis. Using complementation cloning, we have identified a human cDNA that corrects the defect in anchor biosynthesis and allows the expression of glycosylphosphatidylinositol-anchored proteins in Ltk- cells. The nucleotide sequence predicts a novel cytosolic protein of 188 amino acids.

Identification of the region on the class I histocompatibility molecule that interacts with the molecular chaperone, p88 (calnexin, IP90).

During early stages in their biogenesis, murine class I histocompatibility molecules interact transiently with a molecular chaperone of the endoplasmic reticulum designated p88. Using a series of mutant class I heavy chains we mapped the region of the heavy chain that interacts with p88. Domain deletion mutants of the H-2Db and H-2Kb molecules revealed that most of the extracellular portion of the heavy chain and the bulk of the cytoplasmic domain were not required for the association. However, replacement of the transmembrane segment and cytoplasmic domain with a glycosyl phosphatidylinositol anchor from Q7b resulted in a heavy chain that was incapable of interaction with p88. These results suggested that the primary site of interaction with p88 is within a region containing the transmembrane segment and several flanking amino acids of the class I heavy chain. This finding was supported by replacing the glycosyl phosphatidylinositol anchor of the noninteracting Q7b protein with segments of the Db heavy chain containing the putative interaction site and showing that the hybrids were capable of associating with p88. The apparent lack of interaction between segments of p88 and the class I heavy chain that are present within the lumen of the endoplasmic reticulum was also observed when the association between p88 and the alpha chain of the T cell receptor was examined. The full-length transmembrane alpha chain formed a complex with p88, whereas a soluble variant consisting of most of the luminal portion of the alpha chain exhibited only minimal interaction. Thus, p88 is capable of associating with nascent integral membrane proteins through transmembrane interactions that are unavailable to the major soluble chaperone of the endoplasmic reticulum, BiP (GRP78).

Surface transport and internalization of the membrane IgM H chain in the absence of the Mb-1 and B29 proteins.

The micron IgH chain is one component of the membrane IgM complex, the endocytic and signal transducing receptor for Ag on the surface of B lymphocytes. It is transported to the cell surface in association with three other B cell-specific proteins, an L chain and the products of the mb-1 and B29 genes. The roles played by these various proteins in mediating the functions of the complex are unclear. To analyze micron function in the absence of other lymphoid-specific proteins, we first attempted to express micron on the surface of nonlymphoid cells. Deletion of the CH1 domain was sufficient to allow surface expression of this protein in transfected COS cells as well as in mouse L cells. To determine whether this extracellularly truncated micron was capable of endocytosis, we used an assay to detect the internalization of anti-mu antibody bound to the surface of transfected cells expressing the protein. Under both cross-linking and non cross-linking conditions, the CH1-deleted micron protein was internalized in endocytic vesicles. We conclude from these observations that a) the CH1 domain of micron contains a retention signal, the elimination of which allows surface transport of this protein, and b) micron by itself is capable of at least one of the functions of the membrane IgM complex.

Antigenic heterogeneity of Qa-2 antigens in C57BL/6.

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of 125I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.

Glycosyl-phosphatidylinositol-anchored membrane proteins.

Many proteins of eukaryotic cells are anchored to membranes by covalent linkage to glycosyl-phosphatidylinositol (GPI). These proteins lack a transmembrane domain, have no cytoplasmic tail, and are, therefore, located exclusively on the extracellular side of the plasma membrane. GPI-anchored proteins form a diverse family of molecules that includes membrane-associated enzymes, adhesion molecules, activation antigens, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. In the kidney, several GPI-anchored proteins have been identified, including uromodulin (Tamm-Horsfall glycoprotein), carbonic anhydrase type IV, alkaline phosphatase, Thy-1, BP-3, aminopeptidase P, and dipeptidylpeptidase. GPI-anchored proteins can be released from membranes with specific phospholipases and can be recovered from the detergent-insoluble pellet after Triton X-114 treatment of membranes. All GPI-anchored proteins are initially synthesized with a transmembrane anchor, but after translocation across the membrane of the endoplasmic reticulum, the ecto-domain of the protein is cleaved and covalently linked to a preformed GPI anchor by a specific transamidase enzyme. Although it remains obscure why so many proteins are endowed with a GPI anchor, the presence of a GPI anchor does confer some functional characteristics to proteins: (1) it is a strong apical targeting signal in polarized epithelial cells; (2) GPI-anchored proteins do not cluster into clathrin-coated pits but instead are concentrated into specialized lipid domains in the membrane, including so-called smooth pinocytotic vesicles, or caveoli; (3) GPI-anchored proteins can act as activation antigens in the immune system; (4) when the GPI anchor is cleaved by PI-phospholipase C or PI-phospholipase D, second messengers for signal transduction may be generated; (5) the GPI anchor can modulate antigen presentation by major histocompatibility complex molecules. Finally, at least one human disease, paroxysmal nocturnal hemoglobinuria, is a result of defective GPI anchor addition to plasma membrane proteins.

Expression of a transfected H-2Kb gene in B16 cells correlates with suppression of liver metastases in triple immunodeficient mice.

In vivo experiments performed with NIH (nu/nu, bg/bg, xid/xid) triple immunodeficient (TD) mice revealed the striking ability of i.v. injected B16-F1 and B16-F10 murine melanoma cells to colonize not only the lungs but also the liver of TD mice. Subsequently, B16 melanoma cell cultures, which express very low levels of H-2Kb antigen, were cotransfected with plasmids pRSVneo, containing the neomycin resistance gene, and 6-2B1pMT, expressing the H-2Kb complentary DNA under the control of the metallothionein enhancer-promoter. Several neomycin-resistant clones were analyzed for H-2Kb and H-2Db expression by RNase protection and flow cytometry assays. All parental lines and transfected clones expressed normal levels of H-2Db mRNA, while only some of the transfected clones expressed easily detectable levels of H-2Kb mRNA. Moreover, in these clones H-2Kb expression could be enhanced in the presence of Zn2+, indicating that the metallothionein enhancer was functioning properly. Parental cells and transfected clones were injected i.v. in TD mice to assess the possible involvement of H-2Kb antigen in regulating the metastatic potential of B16 melanoma cells. We observed a remarkable correlation between expression of H-2Kb antigen and suppression of liver-specific metastases in TD mice. Identical results were obtained when we gave TD mice injections of mixed populations of transfectants expressing H-2Kb antigen, obtained by fluorescence-activated cell sorting. These experiments allowed us to rule out the possibility that the observed changes in metastatic potential were due to clonal variability among individual transfected clones. Taken together, the results of our in vivo studies with immunodeficient mice support the notion that specific major histocompatibility complex Class I molecules modulate the metastatic potential of malignant cells also by mechanisms which are independent of their well-established role in antigen presentation.

Developmentally regulated polyadenylation of two discrete messenger ribonucleic acids for müllerian inhibiting substance.

Mullerian inhibiting substance (MIS) is a 140-kilodalton homodimeric glycoprotein that causes regression of the Mullerian ducts in male embryos, and may also have a role in both males and females in the regulation of germ cell maturation. We examined the ontogeny of MIS messenger RNA (mRNA) in rat testes from midgestation through adulthood and found two discrete MIS mRNA species that are developmentally regulated. The larger 2.0-kilobase species is abundant at embryonic day 14, then decreases in late gestation, and is barely detectable after birth. The smaller 1.8-kilobase species is first noted at embryonic day 18 and is the major species detected postnatally. Both species are abundant just prior to birth, at embryonic day 21, then decrease markedly after birth. This variation in MIS mRNA levels correlates with the developmental expression of MIS protein. A series of oligonucleotide-directed ribonuclease H mapping experiments determined that the two mRNA species differ at their 3' ends in the extent of polyadenylation. Thus, differential polyadenylation of MIS mRNA may be an additional mechanism for regulating MIS expression during fetal and postnatal development.

The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.

Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.

Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C.

Cell surface antigens thought to be linked to the membrane via phosphatidylinositol (PI) are incompletely, and variably, released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). The basis for this was investigated with cloned tumor cell lines and PI-PLCs isolated from two species of bacteria. Residual Thy-1 antigen, which was detectable by flow cytometry, remained on all thymoma cell lines after exposure to very high concentrations of either purified enzyme. A majority of the presumptive PI anchored molecules on all of the cell lines was sensitive to release by PI-PLC derived from Bacillus thuringiensis. However, cell lines differed dramatically in the ease with which PI-PLC from Staphylococcus aureus liberated the same surface antigens. This heterogeneity was determined at the single cell level because at least five different PI-anchored antigens exhibited similar behavior on a given cell line or transfected subclones of it. The two phospholipases differed with respect to molecular mass, serological cross-reactivity and sensitivity to inhibition by NaCl and detergents. These observations suggest that the two types of PI-PLC may have distinct substrate specificities or sensitivities to environmental conditions which account for the difference in their ability to act on PI-anchored proteins in particular cell types. Such enzymes should continue to be important tools for investigating the method and significance of attachment of lymphocyte surface glycoproteins. In particular, the S. aureus PI-PLC can be used to demonstrate and investigate a previously unrecognized heterogeneity in cells which express PI-anchored molecules.