Hygene Research Articles

Ubiquitary Expressed Major Histocompatibility (MHC) Class I and Minor Histocompatibility Alloantigens (mHAg) Are No Relevant Targets For Graft-Versus-Tumor (GvT) Reactions In Mice After Allogeneic Bone Marrow Transplantation

IntroductionCell mediated GvT effects after allogeneic stem cell transplantation are heterogeneous with respect to effector cell populations, target antigens and their interrelation with graft-versus-host disease (GvHD). Mutated tumor-associated antigens (TAAs), as well as selectively or aberrantly expressed nonmutated antigens, represent potential targets for T cell-mediated GvT effects that are in principle separable from the generalized graft-versus-host reaction targeting widely expressed MHC alloantigens and mHAg. So far, it remains a matter of debate whether alloantigens ubiquitary expressed on tumor cells and normal host tissue are relevant targets of GvT responses. MethodsTo evaluate the role of MHC class I molecules and mHAg as targets for GvT reactions, we employed three different allogeneic parent-into-F1 murine transplant models (BALB/c [H-2d] or C57BL/6 [H-2b] → F1[BALB/c x C57BL/6] [H-2b/d] and BALB/c → F1[BALB/c x CBA/J] [H-2d/k]). As previously shown, these transplant models allow for separate variation in the histocompatibility between donor cells and normal host tissue on the one hand and between donor cells and tumor tissue on the other hand. Test tumors were the donor derived myeloma / leukemia cell lines MPC11 [H-2d] and C1498 [H-2b], which were stably transfected with MHC class I molecules H-2Kb, H-2Kd, H-2Kk or with the mHAg UTY. ResultsCompared with non-GvHD controls (F1[H-2b/d] mice transplanted with allogeneic bone marrow cells from BALB/c [H-2d] alone), significant GvT effects against the BALB/c derived tumor MPC11 occurred in F1[H-2b/d] recipients with severe GvHD (transplanted with bone marrow and additional splenic lymphocytes from BALB/c). Identical histocompatibility antigens of donor and tumor cells precluded allorecognition of tumor cells, leaving TAAs the only possible target antigens in this particular setting, with GvHD as a driving force for augmentation of tumor specific immune responses. The MPC11 myeloma [H-2d], stably transfected with the C57BL/6 derived MHC class I molecule H-2Kb (MPC11-Kb), showed similar tumor growth compared to the wild type MPC11 in F1[H-2b/d] mice transplanted from BALB/c donors. Similar results could be observed with the C57BL/6 derived C1498 leukemia [H-2b], transfected with the BALB/c MHC class I alloantigen H2Kd, in F1[H-2b/d] mice transplanted from C57BL/6 donors. In both experimental settings, the artificially expressed alloantigens H2Kb and H2Kd were also expressed ubiquitarily on tissues of F1[H-2b/d] recipients. In sharp contrast, MPC11 cells transfected with the CBA/J derived H2Kk (MPC11-Kk) alloantigen showed a significantly reduced tumor growth in F1[H-2b/d] recipients transplanted from BALB/c donors, demonstrating that MHC class I alloantigens are relevant targets, when expressed on tumors but not on healthy recipient tissue. Inoculation of MPC11-Kk in F1[H-2d/k] mice 3 or 7 days after transplantation from BALB/c donors showed a trend towards a reduced tumor growth compared to wild type MPC11 in recipients with severe GvHD. Later application of tumor cells (14 days after transplantation) showed similar growth pattern of both tumors, suggesting that MHC class I antigens might be GvT targets in the early phase after allogeneic transplantation. To evaluate the impact of ubiquitarily expressed mHAg, we established a C1498 cell line, which stably expressed UTY (C1498-UTY), a known immunogenic antigen of the male HY gene. In comparison with the wild type control, C1498-UTY showed similar tumor growth in male or female F1[H-2b/d] recipients, transplanted from naive female C57BL/6 donors. Immunization of female C57BL/6 donors with male splenocytes resulted in a significantly reduced tumor growth of the C1498-UTY in female F1[H-2b/d] recipients. In contrast, tumor growth of the UTY expressing and wild type C1498 was similar in male F1[H-2b/d] mice transplanted from immunized female C57BL/6 donors. ConclusionOur experimental data indicate that a biologically relevant GvT effect targeting MHC class I or mHAgs mainly occurs when these alloantigens are not ubiquitarily expressed, even in the context of severe GvHD. Consequently, TAAs and tissue- /hematopoietic specific antigens are most likely the main targets of GvT effects observed after allogeneic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

The case of the midwife scientist.

Genes controlling both testis determining and expression of the male-specific transplantation antigen, HY, are located on the short arm of the mouse Y chromosome, and on the X and Y-linked translocation, Sxr(a). A mutation of Sxr(a) was discovered in a cross between an Sxr carrier male and a T16H/X female. This was designated Sxr(b) and found to affect both the expression of HY and spermatogenesis, but not testis differentiation, thereby disproving Ohno's hypothesis that HY controlled testis determination. Molecular genetic analysis showed the mutation to be caused by fusion of two duplicated genes, Zfy1 and Zfy2, deleting the intervening DNA. This deletion interval, deltaSxr(b), contained a number of genes, each a candidate HY gene. Expression cloning with HY-specific T cell clones identified Smcy, Uty and Dby as encoding peptide epitopes of this transplantation antigen. The human homologues SMCY and UTY likewise express HY antigens and these are targets of damaging graft-versus-host (GVH) responses and potentially therapeutic graft-versus-leukaemia (GVL) responses following bone marrow transplantation (BMT). Knowledge of the peptide identity of HY epitopes allows monitoring of immune responses following BMT, using fluorescent tetramers, and also offers the possibility of inducing immunological tolerance.

Functional identification of genes up- and down-regulated by glucocorticoids in AtT-20 pituitary cells using an enhancer trap.

The AtT-20/D1 mouse pituitary tumor cell line has been used to study glucocorticoid regulation of POMC. We have used an enhancer trap to determine whether other glucocorticoid-regulated genes exist in AtT-20 cells. An enhancer trap is a recombinant construction containing a selectable marker driven by a promoter that has been weakened by removal of its enhancers so that the transfected trap is only expressed if it comes under the influence of an endogenous enhancer. For a selectable marker, we used a fusion gene coding for hygromycin phosphotransferase (Hy) and herpes simplex thymidine kinase. Thus, expression of this gene conferred hygromycin resistance and ganciclovir sensitivity. Suppression resulted in ganciclovir resistance and hygromycin sensitivity. An enhancerless promoter was produced using a truncated, transcriptionally inactive, form of the POMC promoter. AtT-20/D1 cells were transfected with this construct and cultured in medium containing hygromycin to kill any cells not expressing the Hy gene. The survivors were cultured in medium containing ganciclovir and dexamethasone and cloned. Clones in which the transgene was down-regulated by dexamethasone survived and were designated AtT-20/NET (for negative enhancer trap). Northern blot analysis confirmed that the transgene was down-regulated by dexamethasone as expected and that in at least one instance, suppression of the transgene was more complete than suppression of the full-length POMC promoter. Southern blot analysis after restriction enzyme digestion showed that each cell clone contained a single copy of the transgene, and PCR analysis of the promoter region showed that insertion had occurred in two unique sites in at least two cell clones. Another plasmid construct was prepared that contained the selectable gene but lacked any promoter elements. After transfection of AtT-20 cells with this vector, up-regulated enhancers were trapped by selection in hygromycin and dexamethasone followed by ganciclovir alone and designated AtT-20/PET cells (for positive enhancer trap). Up-regulation of the selectable gene in AtT-20/PET cells was confirmed by Northern blot analysis of dexamethasone-treated cells. In summary, glucocorticoid-regulated enhancers have been identified in AtT-20/D1 cells by an enhancer trap strategy that uses sequential selection under conditions that test whether the transgene is active. These results indicate that in addition to the well characterized, down-regulated POMC gene, there are other glucocorticoid-regulated genes in AtT-20/D1 cells that are both up-regulated and down-regulated by glucocorticoids.

Functional identification of genes up- and down-regulated by glucocorticoids in AtT-20 pituitary cells using an enhancer trap

The AtT-20/D1 mouse pituitary tumor cell line has been used to study glucocorticoid regulation of POMC. We have used an enhancer trap to determine whether other glucocorticoid-regulated genes exist in AtT-20 cells. An enhancer trap is a recombinant construction containing a selectable marker driven by a promoter that has been weakened by removal of its enhancers so that the transfected trap is only expressed if it comes under the influence of an endogenous enhancer. For a selectable marker, we used a fusion gene coding for hygromycin phosphotransferase (Hy) and herpes simplex thymidine kinase. Thus, expression of this gene conferred hygromycin resistance and ganciclovir sensitivity. Suppression resulted in ganciclovir resistance and hygromycin sensitivity. An enhancerless promoter was produced using a truncated, transcriptionally inactive, form of the POMC promoter. AtT-20/D1 cells were transfected with this construct and cultured in medium containing hygromycin to kill any cells not expressing the Hy gene. The survivors were cultured in medium containing ganciclovir and dexamethasone and cloned. Clones in which the transgene was down-regulated by dexamethasone survived and were designated AtT-20/NET (for negative enhancer trap). Northern blot analysis confirmed that the transgene was down-regulated by dexamethasone as expected and that in at least one instance, suppression of the transgene was more complete than suppression of the full-length POMC promoter. Southern blot analysis after restriction enzyme digestion showed that each cell clone contained a single copy of the transgene, and PCR analysis of the promoter region showed that insertion had occurred in two unique sites in at least two cell clones. Another plasmid construct was prepared that contained the selectable gene but lacked any promoter elements. After transfection of AtT-20 cells with this vector, up-regulated enhancers were trapped by selection in hygromycin and dexamethasone followed by ganciclovir alone and designated AtT-20/PET cells (for positive enhancer trap). Up-regulation of the selectable gene in AtT-20/PET cells was confirmed by Northern blot analysis of dexamethasone-treated cells. In summary, glucocorticoid-regulated enhancers have been identified in AtT-20/D1 cells by an enhancer trap strategy that uses sequential selection under conditions that test whether the transgene is active. These results indicate that in addition to the well characterized, down-regulated POMC gene, there are other glucocorticoid-regulated genes in AtT-20/D1 cells that are both up-regulated and down-regulated by glucocorticoids.

A Case Report of Maxillofacial Prosthesis on 7 Cases

As compared with general prosthodontic treatment, maxillary prosthetics is difficult to make a plan, treatment, and postoperative control, so that it has large defects and denture bearing area decreases, the changing quantity of defective bone is large is the course of time.This time we experienced the 7 cases of disease (4 upper cases, 3 lower cases) including 5 cases by tumour, 1 case by trauma, and 1 case by cyst.In case of maxilla, we treated on prosthodontics after radical operation, in case of mandible, we made it after radical operation, recover operations for example implantation for iliac bone dermatoplasty.As shown under, we know that conclusions we cured defective parts of bomy structured by obturator in case of maxilla chiefly we use the system of bar clasp for remained teeth to make maintenance and stability.In aim of support, we used flange technique to improve border molding, and used Levin's bladed metal teeth to relieve the mucous charge's quantity by force of bite and keep out of eccentric moments.Meanwhile, it is difficult for mandible to recover by denture in case of having the missing bomy parts.Therefore we made prothesis, after implantation of iliac bone, and dermatoplasy, improvement of discrepancy as well as we could in mandible.The plan of plate in the case like this was carried out, not only in the view of anatomy, but also in the point of dynamics to disperse force of bite well and balance square of implantations with original own jaw's bone.And in case which we experience so that we could use residual teeth, we use Dolder's bar attachment in order to be lower the point of force and splint of residual teeth for retainer of denture.The prognosis of all cases were improved on physiologic functions and esthetics.Especially the 7th cases, though it passed ten years after having denture, the mobility of abutment teeth resorption of transplantationed bone can not be observed and oral hygene condition is good and make various physiologic functions.