Survival of cultured allografts in patients with burns assessed with probe specific for Y chromosome.

The aim of the study was to determine the fate of cultured skin allografts in patients with burns. In situ DNA hybridisation with a Y probe (pHY 2.1) was used to detect cells carrying the Y chromosome (the probe being visualised by the alkaline phosphatase-antialkaline phosphatase method) in biopsy specimens taken from cultured allografts derived from donors of the opposite sex to the recipients (20 patients with burns). Specimens were taken within a week, between one and three weeks, between four and six weeks, and more than six weeks after grafting. Only two of the 27 biopsy specimens contained cells that were the same sex as the donor; both were taken within a week after grafting. In the 25 other specimens the epithelial cells were the same sex as the recipient. Cultured skin allografts showed no evidence of survival in patients with burns, which suggests that they are probably not suitable for long term management of burns but may be useful as short term biological dressings.     

Use of allograft biopsies to assess thymopoiesis after thymus transplantation

Thymus allograft biopsies were performed in athymic infants with complete DiGeorge anomaly after thymus transplantation to assess whether the thymus allograft tissue was able to support thymopoiesis. Forty-four consecutive infants were treated with postnatal cultured thymus allografts. Thirty biopsies and six autopsies evaluating the allograft site were obtained in 33 infants, 23 of whom survive. The allograft was examined by immunohistochemistry for evidence of thymopoiesis. Grafted thymus tissue was found in 25 of 30 biopsies, 23 of which showed thymopoiesis. All 19 survivors with thymopoiesis on biopsy developed naive T cells and T cell function. Autopsies were done in six subjects, three of whom had biopsies. All autopsy samples contained thymus tissue including one for which the biopsy had not contained graft. Of the six autopsies, one had evidence of thymopoiesis. Epithelium without thymopoiesis was seen in two of 25 biopsies in which thymus tissue was detected and in five of six autopsies. Graft rejection was seen in one autopsy. Biopsies were important for showing the following: 1) the damaging effect of pulse steroids on thymopoiesis; 2) the need for adequate immunosuppression of atypical subjects; and 3) the presence of thymopoiesis in the presence of ongoing immunosuppression. In addition, the biopsy could rule out graft rejection in the atypical subjects who had oligoclonal T cells that could cause rejection. In summary, combining biopsy and autopsy data, allogeneic thymus tissues showed thymopoiesis in 24 of 29 (86%) evaluable transplants. The results of these biopsies led to improved care of these complex patients.     

Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis

Human keratinocytes isolated from a skin biopsy and cultured in vitro reconstitute a stratified squamous epithelium suitable for grafting on burned patients. Melanocytes coisolated from the same skin biopsy also proliferate under these culture conditions and maintain differentiated functions (i.e., synthesize melanin granules, regularly intersperse in the basal layer of the cultured epidermis, and transfer melanosomes in the cytoplasm of contiguous keratinocytes) (De Luca, M., A. T. Franzi, F. D'Anna, A. Zicca, E. Albanese, S. Bondanza, and R. Cancedda. 1988. Eur. J. Cell Biol. 46:176-180). Isolated melanocytes in culture grow in the presence of specific growth factors with a mean population doubling time of 4-10 d. In this paper we show that (a) human keratinocytes and oral epithelial cells possess strong and specific melanocyte growth stimulating activity (doubling time, 24 h); (b) melanocyte growth is not autonomous but requires close keratinocyte contact and is regulated to maintain a physiological melanocytes/keratinocytes ratiol and (c) pure skin keratinocytes, but not oral epithelial cells, have all the information required for the proper physiological location and differentiation of melanocytes in the epidermis.     

Immunological selection of HL-A variants of cultured progeric fibroblasts and their identification by quinacrine fluorescence

HL-A2 deficient clones of cultured fibroblasts from a boy with progeria were selected immunologically from a thousand-fold excess of HL-A2 positive normal female cells in mixed cultures. Identification of surviving male cells was both confirmed and facilitated by quinacrine fluorescence of the Y chromosome in metaphase plates within several clones. These results support previous findings that progeric cells have altered HL-A antigens and also provide a more generally applicable method of selection for genetic studies on cultured somatic cells.     

Characterization of glycogen phosphorylase isoenzymes present in cultured skeletal muscle from patients with McArdle's disease.

Muscle biopsy specimens from patients with McArdle's disease lack glycogen phosphorylase activity. Significant phosphorylase activity was detected in cultured muscle cells from these patients. The phosphorylase isoenzymes in the cells were identified electrophoretically and immunochemically. On polyacrylamide disc gel electrophoresis, two types of isoenzymes were separated in about equal amounts. Both differed the muscle type in migration, kinetic, and immunochemical properties. The first type corresponded to a fetal phosphorylase isoenzyme, and the second was a liver-like type which was completely absorbed with antibody against the rat liver isoenzyme. No adult skeletal muscle isoenzyme was detected.     

Repigmentation of vitiliginous skin by cultured cells

Epidermal cells from pigmented areas of a patient with vitiligo were cultured in MCDB-153 medium, which supports the clonal growth of undifferentiated keratinocytes and melanocytes. The cells were grown on collagen-coated substrata. After the cells reached semiconfluence, the composite of substratum and cells was emplaced onto dermabraded vitiliginous areas as a graft. Re-epithelialization of the grafted areas was complete after 2 weeks. Repigmentation was evident after 1 month and continued over the observation period of several months. There was complete and normal differentiation of the graft, including a normal distribution of melanocytes in the basal layer. Ultrastructural studies showed a normal distribution of melanosomes in the melanocytes and showed keratinocytes that were indistinguishable from the uninvolved skin.     

Cellular survival in rat vein-to-artery grafts

Microsurgical models of vein-to-artery graft surgery have been developed in rats as a means of assessing vein graft adaptation and neo-intimal hyperplasia. Neo-intimal hyperplasia in these grafts is often attributed, at least in part, to an adaptive response by venous smooth muscle cells to the increased intraluminal pressure of the arterial pressure. However, considerable evidence suggests complete or near-complete cellular replacement in these grafts. A series of experiments were undertaken in which male vein or artery grafts were placed into either allogeneic female nude rat hosts or into syngeneic WKy female hosts as a means of determining donor cell survival. Grafts were removed at postsurgery week 2 or week 6 and the fate of the donor male cells assessed by PCR amplification of the testis-determining gene Sry. The Sry gene was undetectable in 2-week male to female vein grafts. When left for 6 weeks, donor cells were detectable in vein grafts only after multiple 50-cycle PCR analyses. Minimal donor cell survival was not due to an allograft response, as donor male cells were readily detectable in WKy male to female nude rat artery-to-artery grafts. These data were not nude rat specific, as poor donor cell survival was also evidenced in syngeneic male to female vein-to-artery grafts. In conclusion, we demonstrate only marginal survival of donor cells in rat vein-to-artery grafts. Neo-intimal hyperplasia in these grafts was not a consequence of donor venous smooth muscle cell proliferation.     

Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects.     

Frozen cultured sheets of human epidermal keratinocytes enhance healing of full-thickness wounds in mice

Sheets of cultured allogeneic human keratinocytes have been used for the treatment of burns and chronic leg ulcers but there has been no animal assay for the therapeutic action of these cultures. In order to analyze the effects of frozen cultures of human keratinocytes on wound healing, we have developed such an assay based on the rate of repair of full-thickness skin wounds in immunocompetent NMR1 mice. Reepithelialization of the control wounds, originating from the murine epithelium at the edge of the wound, occurred at a constant rate of advance of 150 microm/day. When frozen cultured human epidermal sheets were thawed at room temperature for 5-10 min and applied to the surface of the wound, the murine epithelium advanced at 267 microm/day. Most wounds treated with frozen cultures completely healed after 10 days, whereas most control wounds required 16 days. The accelerated reepithelialization did not depend on the presence of proliferative human keratinocytes in the frozen cultures. The cultures also promoted early formation of granulation tissue and laminin deposition over the surface of the wound bed. This simple assay should permit quantitative analysis of the effects on healing exerted not only by cultured cells, but also by proteins and small molecules.     

Differential frequency of human X and Y chromatin as related to cell density in vitro

The relationship between cell density in vitro and the frequency of fluorescent X chromatin and/or Y chromatin-positive interphase nuclei was studied in cultured cells from three human male and female subjects. It was found that altough the frequency of fluorescent X chromatin-positive cells was proportional to cell density, the frequency of fluorescent Y chromatin-postitive cells was independent of cell density. Our results are compared with those of other investigators.     

解脲脲原体药物敏感性变迁研究

目的:探讨上海地区2008与2012年泌尿生殖道解脲脲原体(Ureaplasma urealyticum,UU)耐药性变化,为临床合理用药提供参考。方法:采用自制UU药物敏感检测试剂对2008年450例和2012年459例患者的标本进行检测,观察UU阳性情况及UU对交沙霉素、氧氟沙星、阿奇霉素和强力霉素的药物敏感性。结果:2008年分离到150例UU阳性标本和2012年分离到134例UU阳性标本分别对交沙霉素、强力霉素、氧氟沙星和阿奇霉素等4种抗菌素的敏感率有显著性差异;2008年46例和2012年38例男性患者阳性标本中分离的UU分别对交沙霉素和阿奇霉素敏感率有显著性差异;对强力霉素和氧氟沙星敏感率无显著性差异。2008年104例和2012年96例女性患者阳性标本中分离的UU分别对交沙霉素、强力霉素、氧氟沙星和阿奇霉素等4中抗菌素的敏感率都有显著性差异。结论:5年间UU药物敏感性发生了变迁;临床医师应该关注本地区药物敏感性变迁,合理选用抗生素。     

Epidermal stem cells and ex vivo cutaneous gene therapy: application to xeroderma pigmentosum

Ex vivo cutaneous gene therapy is an alternative treatment for recessively inherited diseases with cutaneous traits. It relies on the transfer in cultured epidermal keratinocytes of the wild-type allele of the gene whose mutation is responsible for the disease. As for severely burnt patients, epithelial sheets developed from genetically corrected cells may then be grafted back to the patients. Long term correction and graft take depend on the genetic correction of stem cells. Success of such an approach has recently been reported in the case of one patient suffering from a severe case of junctional epidermolysis bullosae. Here we report a method for safely selecting keratinocytes populations after genetic manipulation. The method is non invasive and non immunogenic and allows high enrichment of genetically manipulated stem keratinocytes. This could perhaps contribute to ex vivo gene therapy approaches of cancer prone genodermatoses such as xeroderma pigmentosum.     

Fate of donor cells in vascularized bone grafts: identification of systemic chimerism by the polymerase chain reaction

Systemic chimerism, or the movement of cells from a transplanted tissue into host organs, is a phenomenon known to occur in association with development of immunological tolerance in allotransplantation. However, little is known about the fate and movement of cells into or out of autogenous free tissue transfers, including vascularized bone grafts. The purpose of this study was to identify systemic chimerism in vascularized bone grafts by transplantation of a vascularized tibiofibular graft from isogenous (inbred) male Lewis rats to female recipients. Donor (male) cells could be identified in the recipient (female) tissues by semiquantitative polymerase chain reaction analysis for a Y chromosome-specific DNA sequence. Chimerism was assessed at 1, 12, 18, and 24 weeks after transplantation. Competitive polymerase chain reaction study using the specific primers for a Y-chromosome marker ( gene) and an autosomal gene (GAPDH) allowed detection of small amounts of male cells in a large pool of female cells and measurement of their relative proportions as a function of time. Of 19 nonimmunosuppressed recipients, nine animals (47 percent) showed low-level chimerism (<0.1 percent) in the peripheral blood. Nine (47 percent), three (16 percent), and two (11 percent) recipients showed high-level chimerism (>1 percent) in the spleen, liver, and thymus, respectively, at final assessment. Donor cells were detected in all bone grafts and in six contralateral tibial bones (i.e., 67 percent of sampled contralateral tibial bones) at 18 and 24 weeks after transplantation. Twenty-four recipients were immunosuppressed with FK506 (tacrolimus) to suppress reaction to a minor histocompatibility barrier present on the Y chromosome. In this group, 14 animals (58 percent) showed low-level chimerism in peripheral blood and 12 (50 percent), eight (33 percent), and one (4 percent) recipients showed high-level chimerism in the spleen, thymus, and liver, respectively. Transplanted cells were detected in nine contralateral tibial bones (i.e., 60 percent of sampled contralateral tibial bones) at 12 and 18 weeks after surgery. The results indicate that polymerase chain reaction for the Y chromosome is a useful tool for differentiating between donor and recipient cell populations experimentally using sex-mismatched tissues in a rat model. This study demonstrated that systemic chimerism occurs after successful vascularized bone transplantation. Transplanted cells not only survive in the graft but also gradually migrate into the recipient's body.     

Expression of Ia antigen on epidermal keratinocytes after the grafting of normal skin to nude mice

During the course of experimentation designed to evaluate the migration of host Langerhans cells (LC) into normal skin grafted onto nude mice, we observed that the epidermal cells of these grafts were induced to express la determinants solely of graft origin. The data presented herein indicate that the expression of la by normal epidermal cells correlates with the infiltration of host LC into the graft. This la expression is restricted to the keratinocytes within the epidermis of the grafted skin, is first observed 7 to 9 days post-grafting, and persists within the grafted skin for greater than 12 wk. The induction of la expression by keratinocytes appears to be the result of the environment that is provided by the nude mouse host and is independent of both passenger lymphocytes within the skin graft and allogeneic differences between graft and host. We strongly believe that these studies provide the basis for the development of an experimental animal model system for investigating the potential role that la expression by epidermal cells may play in enhancing the immune response to antigens encountered in the skin.     

Cytomegalovirus Infection of the Cervix Detected By Cytology and Histology: A Report of Five Cases

We present four cases of cytomegalovirus (CMV) infection detected by cervical cytology of asymptomatic young women. One patient who had undergone bone marrow transplantation was immunosuppressed, but no factors predisposing to CMV infection were identified in the other three patients. A cervical biopsy specimen from a fifth patient, who was also asymptomatic, demonstrated the locus of CMV infection to be in endocervical gland cells. Immunocytochemical and polymerase chain reaction studies on this biopsy specimen confirmed that the histological changes were caused by CMV. the finding of CMV-infected cells in cervical cytological or biopsy specimens is a rarity. Our observations in asymptomatic, non-immunocompromised women suggest their presence is an incidental finding, unlikely to have any clinical significance.     

FISH and PCR analyses in three patients with 45,X/46,X,idic(Y) karyotype: clinical and pathologic spectrum

OBJECTIVE: To delineate the phenotypic spectrum (clinical and gonadal features) from patients with a 45,X/46,X,mar(Y) karyotype based upon of their clinical, histological, cytogenetic and molecular evaluation. SUBJECTS: Three patients with a 45,X/46,X,mar(Y) karyotype. METHODS: Clinical assessment, karyotyping, endocrine evaluation, FISH and PCR analyses of several Y-chromosome loci and direct sequencing of the SRY gene. RESULTS: The patients, two males and one female had varying degrees of impairment of sexual differentiation, with or without testis formation. One patient (reared as female and aged 17 years) had Turner syndrome with bilateral streak gonads. The second patient (2.4 years old) had ambiguous genitalia and presented a dysgenetic testis with a contralateral streak gonad. A third patient (26 years old) had bilateral dysgenetic testes (dysgenetic male pseudohermaphroditism). The ratio of 45,X vs. 46,X,+mar(Y) cells differed between patients and between different tissues. In each case the marker sexual chromosome was identified as a rearranged Y-chromosome (idic(Y)) using FISH and PCR analyses. In all cases the SRY gene was present in all tissues studied. No mutations were identified in this gene in any of the patients. CONCLUSIONS: The extent of male or female differentiation in these patients depends in part on the prevalence, time occurrence, and distribution of the 45,X cell line.     

Humoral immune response to melanoma-associated membrane antigen and fetal brain antigen demonstrated by indirect membrane immunofluorescence. I

Summary A melanoma-associated membrane antigen and a fetal brain antigen were identified on the surface of a human melanoma cell line by indirect membrane immunofluorescence techniques. The target melanoma cells were grown in gamma globulin-depleted human serum. Sera from melanoma patients were used as the source of antimelanoma antibodies. To remove alloantibodies, the allogeneic sera were preabsorbed with cultured lymphoblastoid cells derived from the peripheral lymphocytes of the donor of the target cell line. To further define the antigen responsible for antibody activity, sequential absorption tests were performed with fetal brain cells, cultured sarcomas, and breast carcinomas. Some antibody activity was removed by fetal brain tissues. Further absorption with fetal brain or the cultured sarcoma or breast carcinoma did not remove additional activity. However, antibody activity was completely removed by either cultured or biopsy-derived melanoma cells. A serum autochthonous to the target cell line was also tested. The antibody titer of the serum was completely removed by absorption with either autochthonous biopsied tumor or an allogeneic melanoma cell line, but not with the normal tissues. Thus it appeared that sera from melanoma patients contained antibody to both a melanoma-associated membrane antigen and a fetal brain antigen.     

Biological aspects of limb transplantation: I. Migration of transplanted bone marrow cells into recipient

The transplanted limb contains bone marrow tissue. The hematopoietic cells contained in the bone of the graft normally differentiate after transplantation and can be released to the recipient. The cells migrate to the recipient bone marrow cavities and lymphoid organs. This causes the immune reaction between the donor and the recipient, which develops not only in the graft itself but also in the recipient immune organs where donor bone marrow cells home. The purpose of this study was to investigate the process of migration of the hematopoietic cells from the donor limb to the recipient bone marrow cavities and lymphoid tissues. The questions the authors asked were: what is the rate of release of bone marrow cells from the transplanted bone, where do the released bone marrow cells home in the recipient, how fast are donor bone marrow cells rejected by the recipient, and can some bone marrow cells homing in the recipient tissues survive and create a state of microchimerism. Experiments were performed on Brown Norway and Lewis inbred rat strains (n = 30). Limb donors received intravenous chromium-51-labeled bone marrow cells. Twenty-four hours later, the limb with homing labeled bone marrow cells was transplanted to an allogeneic or syngeneic recipient. The rate of radioactivity of bone marrow cells released from the graft and homing in recipient tissues was measured after another 24 hours. To eliminate factors adversely affecting homing such as the "crowding effect" and allogeneic elimination of bone marrow cells by natural killer cells, total body irradiation and antiasialo-GM1 antiserum were applied to recipients before limb transplantation. In rats surviving with the limb grafts for 7 and 30 days, homing of donor bone marrow cells was studied by specific labeling of donor cells and flow cytometry as well as by detecting donor male Y chromosome. The authors found that transplantation of the limb with bone marrow in its natural spatial relationship with stromal cells and blood perfusion brings about immediate but low-rate release of bone marrow cells and their migration to recipient bone marrow and lymphoid tissues. Large portions of allogeneic bone marrow cells are rapidly destroyed in the mechanism of allogeneic elimination by radioresistant but antiasialo-GM1-sensitive natural killer cells. Some transplanted bone marrow cells remain in the recipient's tissues and create a state of cellular and DNA microchimerism. A low number of physiologically released donor bone marrow cells do not seem to adversely affect the clinical outcome of limb grafting. Quite the opposite, a slight prolongation of the graft survival time was observed.     

H-Y antigen: No evidence for alleles in wild strains of mice

H-Y antigen(s) coded or controlled by the Y chromosome in a variety of wild mouse strains have been compared with those of the inbred laboratory strains C57BL/6 (B6) and C57BL/10 (B10). H-Y antigen(s) were detected by H-2-restricted cytotoxic T cells from B6 and B10 female mice primed in vivo and boosted in vitro with syngeneic male spleen cells: There was no difference in the degree of H-Y specific lysis of male cells from the C57BL strains and of F₁ hybrids or B6 congenic mice carrying the Y chromosome from the wild mouse strains examined. This result indicated that at the level of target cell specificity the H-Y antigen(s) from wild and laboratory strains were indistinguishable. H-Y antigen(s) were also found to be indistinguishable at the level of the in vitro induction of the anti H-Y cytotoxic response: F₁ female mice, primed in vivo and boosted in vitro with homologous F₁ male cells, all made H-Y-specific responses and where it could be examined, the target cell specificity of the anti-H-Y cytotoxic cells showed that B10 male cells as well as the homologous F₁ male cells (where the Y chromosome was derived from the wild strain) were good targets. Finally, possible differences in H-Y transplantation antigens between the wild strains and the B10 laboratory strain were examined by grafting F₁ male mice, the progeny of B10 females, and wild strain males with B10 male skin. These grafts were not rejected during an observation period of more than 9 months. Taken together, neither the cytotoxic data nor the skin graft data provide any evidence for allelism of H-Y even though the mouse strains examined were collected from widely disparate geographical locations.