Association of MYH9 rs3752462 and rs5756168 Polymorphisms With Transplanted Kidney Artery Stenosis.

Allelic variants of the MYH9 gene, encoding myosin nonmuscle heavy chain type IIA, have been shown to correlate with diminished glomerular filtration rates and end-stage kidney disease in individuals of Caucasian ancestry. Myosin nonmuscle heavy chain type IIA is expressed during development as well as in injured vessels and kidney structures. We hypothesized that MYH9 risk variants may correlate with kidney artery injury and dysfunctional healing, such as transplant renal artery stenosis (TRAS). Our study aimed at evaluating the association of MYH9 risk allelic variants (rs4821480, rs4821481, rs3752462, rs11089788, rs136211, rs5756168, rs2032487, and rs2239784) with TRAS, defined as >50% renal artery lumen reduction. Genotyping was performed with the use of custom Taqman genotyping assays on DNA samples (n = 295) from white deceased-donor kidney transplant recipients and genomic DNA from the corresponding donors. Statistical analysis was performed with the use of Kaplan-Meier estimates, log-rank tests, and proportional hazard Cox models. Recipients carrying TT in rs5756168 experienced diminished risk of TRAS (hazard ratio [HR], 0.31; P < .009), whereas organs carrying CC in rs3752462 were exposed to excessive TRAS risk (HR, 2.54; P < .047). In multivariate stepwise analysis TRAS was 10.9-fold increased in kidneys originating from rs3752462 CC, whereas the risk was decreased 3.45-fold (adjusted HR, 0.29) in recipients carrying rs5756168 TT (P < .007 and P < .033, respectively). Intracranial bleeding or trauma compared with other mechanisms of donor death diminished TRAS risk by 87% and 91%, respectively (P < .030 and P < .017). Our study is the first to identify genetic predisposition to transplant renal artery stenosis.

Comparison of bone formed intramuscularly after transplantation of scapular and calvarial osteoblasts.

Previous work suggested that osteoblasts determine the size of the bone marrow area within the bone and that calvarial osteoblasts differ from those induced intramuscularly by cartilage formed by transplanted epiphyseal chondrocytes. This study reports morphological observations of bone formed by transplanted scapular and calvarial osteoblasts isolated from bones of young rats. In intact scapulas of 28-day-old rats the percentage area occupied by bone tissue in relation to bone marrow was 6 times larger than in parietal bones of comparable age. Isolated syngeneic scapular osteoblasts usually produced an ossicle with similar general structure and ratio bone tissue/bone marrow area as in intact scapulas. In transplants of calvarial osteoblasts numerous islands of bone tissue with a small amount of bone marrow appeared. Bone formed in allogenic transplants was rejected. These results suggest that osteoblasts from endochondral scapular bone may have different properties than those from intramembranous calvarial bones. Alternatively, the large amount of medullary space in bone produced by transplanted scapular osteoblasts could result from their contamination with bone marrow stromal cells.

Rejection of cartilage formed by transplanted allogeneic chondrocytes: evaluation with monoclonal antibodies.

Cellular infiltrates participating in rejection of cartilage formed by transplanted allogeneic rat epiphyseal chondrocytes were evaluated immunohistochemically using a panel of different monoclonal antibodies. One week after transplantation, the grafts were surrounded by numerous class II MHC+ (OX6+, OX17+), CD4+ (W3/25+), and W3/13+ cells as well as some ED1+ monocytes/macrophages. Only a few T (OX19+) and B (HIS14+) cells were present. The number of class II MHC+ cells and ED1+ monocytes/macrophages did not change significantly in the course of rejection whereas the number of CD4+ and W3/13+ cells gradually decreased. On the other hand, there was a significant increase in the number of CD8+ (OX8+) cells. CD8+ cells accumulated close to the transplants and some of them penetrated cartilage matrix suggesting that they might be involved in chondrocyte killing. After 3 months, cartilage was almost completely destroyed and the intensity of infiltrations was markedly decreased. Fibrous connective tissue predominated, however, some class II+ as well as few ED1+, CD4+ and CD8+ cells were still present adjacent to the cartilage remnants. At the time of transplantation, chondrocytes were endowed with RT1.D class II antigen (OX17+), but they did not react with OX6 mAb (monoclonal antibody) recognizing the RT1.B class II molecule. However, after 1 week, some chondrocytes reacted with OX6 mAb and the number of RT1.B positive chondrocytes increased in the course of cartilage rejection.

Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats.

Rat chondrocytes isolated from the articular-epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte-chondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.

Differences in cartilage formed intramuscularly or in joint surface defects by syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex.

Syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex were suspended in hyaluronic acid and transplanted intramuscularly or into joint surface defects. Transplants were fixed in ruthenium hexammonium trichloride and embedded in glycol methacrylate. In cartilage nodules produced intramuscularly, chondrocyte hypertrophy and matrix calcification were observed after 2 wk. Partial ossification occurred after 4 wk and the cartilage was almost completely replaced by an ossicle after 8 wk. Only small, dispersed groups of chondrocytes remained within the ossicle. In cartilage formed in joint surface defects a superficial and a deep zone were distinguished. Chondrocytes in the superficial zone did not hypertrophy and cartilage remained unossified. In the deep zone matrix calcification and bone formation occurred. These processes were, however, retarded in comparison with intramuscular transplants. Thus, either intraarticular environment exerted an inhibitory effect on chondrocyte hypertrophy and matrix calcification or articular chondrocytes present among transplanted cells accumulated close to the joint lumen and reconstructed normal articular cartilage.

Potential application of cytokine level measurement in corneal epithelium.

The aim of the work was to evaluate whether rat corneal epithelial (RtCE) cell line, spontaneously established from rat corneal epithelium in our laboratory, could be used for the evaluation of cornea inflammatory state. Production of proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha by RtCE line in response to a non-specific irritating agent (Triton) was tested. Supernatants from RtCE cells treated for 1 h with 20 microM, 50 microM and 100 microM Triton, were collected after 1 and 24 h, and tested with enzyme-linked immunosorbent assay (ELISA) for IL-1beta, IL-6 and TNF-alpha. The control groups did not produce significant levels of any of the cytokines. However, after stimulation with Triton, the cells did not produce TNF-alpha, while the concentration of IL-1beta and IL-6 increased over 10 times. These results show that in response to a proinflammatory agent RtCE line produces cytokines that could be used for measuring the effect of irritants on the cornea.

Clinical aspects of disrupted Hedgehog signaling (Review).

The Hedgehog (HH) signaling pathway is involved in patterning and development of a variety of organ systems, including the nervous system, the skeletal system, the craniofacial structures, and the gastrointestinal tract. Recent evidence also implicates this signaling pathway in the postembryonic regulation of stem-cell number in epithelia and blood. The family of HH proteins consists of at least three different members, i.e., sonic HH (SHH), Indian HH (IHH), and desert HH (DHH). SHH is the most broadly expressed member of this family and is probably responsible for the major effects of this signaling pathway. The HH signal is received and transduced via a specific receptor complex composed of patched (PTCH) and smoothened (SMOH) transmembrane proteins. Abnormalities in this signaling cascade have been found in various developmental pathologies and neoplasms such as basal cell carcinoma. The abnormalities are associated with congenital or sporadic genetic alteration affecting function of different components of the HH signaling pathway, including SHH, PTCH, SMOH and GLI proteins.

Cartilage transplants in normal and preimmunized mice.

Syngeneic, H-Y incompatible and allogeneic rib and nasal cartilages from 5-day- and 8-week-old mice were transplanted into adult recipients. Some recipients of allogeneic transplants were preimmunized with splenocytes or chondrocytes from animals of the same strain as cartilage donors, and some with sheep red blood cells (SRBC). Syngeneic or allogeneic transplants independently of the age of the cartilage, did not evoke any detectable humoral or cellular response as judged by evaluation of the specific cytotoxic antibodies and indirect migration inhibition test, respectively. Allogeneic transplants of nasal cartilage were free of infiltrating lymphoid cells, but in the vicinity of rib cartilage grafts some lymphocytes were present. Animals preimmunized with allogeneic splenocytes or chondrocytes displayed both humoral and cellular response to donor cells. Allogeneic cartilage transplants from these animals were surrounded by heavy infiltrations. Preimmunization with SRBC did not evoke infiltrations around allogeneic cartilage grafts.

Organ and age distribution of natural anti-chondrocyte cytotoxicity in the mouse.

Natural cell-mediated cytotoxic activity of mouse lymphoid cells against isolated epiphyseal chondrocytes was tested in 18 h chromium-release assay. The highest anti-chondrocyte cytotoxicity was demonstrated with spleen- and peritoneum-derived leukocytes. Bone marrow cells and lymph node cells exerted weak activity while thymocytes and Peyer's patch-derived cells exerted no cytotoxic activity. Slight, but significant, activity appeared in spleen of 1-week-old mice. It increased with age reaching the maximum in about the 8th week which was followed by subsequent decrease. The ability to lyse isolated chondrocytes by peritoneal cells appeared in 3-6th week of life. It reached the maximum on about the 8th week and disappeared completely in about the 12th week. On the contrary, there were no changes in anti-chondrocyte activity of bone marrow cells.

Immunogenicity of allogeneic pancreatic islet grafts: the effect of in vitro culture and the site of transplantation.

Freshly isolated or cultured for 14 days rat allogeneic pancreatic islets were transplanted either intramuscularly or intratesticularly. After 14 days, the specific recipients' antigraft humoral and cellular immunity evaluated by means of leukoagglutination assay and indirect splenocyte migration inhibition test, respectively. Islet preculture resulted in significant decrease of humoral and in absence of cellular antigraft immunity irrespectively of site of transplantation. Intratesticular grafting did not evoke cellular response after transplantation of both fresh and cultured islets. However, antibody production after intratesticular transplantation of fresh islets remained unaffected. It may be concluded that preculture of rat pancreatic islets affects their immunogenicity. Moreover, testis appears to be a suitable immunoprivileged site for islet grafting.

Augmented pro-apoptotic effects of TRAIL and proteasome inhibitor in human promonocytic leukemic U937 cells.

TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand), a member of the TNF family, is known to be cytotoxic for a high proportion of tumor cell lines. However, successful application of TRAIL in tumor therapy may depend on finding other agents that can potentiate its antitumor effects. The present study showed that the cytostatic/cytotoxic TRAIL activity against U937 cells could be significantly augmented by proteasome inhibitor PSI, as revealed by MTT assay. Increased cytostatic/cytotoxic effect on U937 cells by TRAIL/PSI combined treatment was caused by apoptosis, as shown by an increased PARP cleavage rate. TRAIL/PSI did not affect the level of mRNA expression for TRAIL receptors (DR4, DR5, DcR1) and other apoptosis signal transduction molecules (TRADD, caspase-8).

Synergistic antiproliferative activity of tumor necrosis factor alpha (TNF-alpha) and lovastatin.

We assessed the antiproliferative effect of tumor necrosis factor alpha (TNF-alpha) and lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, alone and in combination, on two murine tumor cell lines. Recombinant TNF-alpha inhibited proliferation of murine MmB16 melanoma cells in a concentration-dependent fashion but stimulated growth of murine L1210 leukemia cells at 0.1 ng/ml concentration. Lovastatin inhibited proliferation both of murine MmB16 melanoma cells and of murine L1210 leukemia cells in a concentration-dependent fashion. In combination with tumor necrosis factor alpha lovastatin inhibited synergistically growth of both cell lines as assessed by isobologram analysis. Our data show that lovastatin, a cholesterol synthesis inhibitor, introduced to the clinic to treat hypercholesterolemia, used either as a single or in combination with TNF-alpha inhibits growth of MmB16 melanoma and L1210 leukemia cells.

Structural differences between bone formed intramuscularly following the transplantation of isolated calvarial bone cells or chondrocytes.

Bone formed in intramuscular transplants of isolated syngeneic calvarial bone cells in mice, was compared with endochondral bone induced by cartilage produced by analogous transplants of isolated epiphyseal chondrocytes, as well as with parietal bones forming the bulk of the calvaria. Transplanted calvarial cells produced islands of bone, some of which contained intraosseous cavities. Osteoclasts inside these cavities were observed only in 14-day-old transplants and bone marrow cells in 28-day and older transplants. On the contrary, bone marrow appeared soon after formation of bone trabeculae in endochondral bone. The percentage area occupied by bone marrow in these specimens was about twentyfold larger than in the bone formed by transplanted bone cells. On the other hand, the bone marrow area in the latter type of bone was somewhat smaller but of similar order as in parietal bones. Moreover, both in parietal bones and in bone formed by isolated bone cells, the bone marrow was devoid of fat cells which were numerous in bone arising by endochondral ossification. It appears, therefore, that the ratio of bone marrow to the bone tissue area in parietal bones depends more on the intrinsic properties of osteoblasts than on the local factors in the environment of the developing bone. In the case of bone induced by cartilage, the bone marrow/bone tissue area could be determined both by the extent of cartilage resorption by vascularized tissue and by the properties of osteoblasts.

Difference in shape of bone formed by isolated calvarial and scapular osteoblasts transplanted under various conditions.

To study the influence of transplantation conditions on early stages of osteogenesis, isolated calvarial or scapular osteoblasts were injected into the leg or dorsal muscles (free transplants) or implanted after seeding on fragments of devitalized parietal bones (supported transplants) into dorsal muscles. The cross-sections of bone islands formed by calvarial osteoblasts in the different types of transplants were then compared according to their maximal breadth and length. Moreover, the same dimensions of pieces of bone formed by scapular osteoblasts in supported transplants were compared with those of bones formed in free transplants into leg muscles. Finally, comparison of the dimensions of cross-sections of supported transplants of calvarial and scapular osteoblasts was done. Calvarial osteoblasts in dorsal muscles produced a slightly higher percentage of wider and longer islands than those in leg muscles. In supported transplants of calvarial osteoblasts the percentage of narrow bone islands (breadth less than 100 microns) was considerably higher than in free transplants. Similarly, the percentage of narrow cross-sections in bones formed by scapular osteoblasts was higher in supported than in free transplants. In supported transplants of calvarial osteoblasts the percentage of narrow islands was higher than in similar transplants of scapular bone cells. It is suggested that the differences in shape of pieces of bone formed in supported and free transplants reflect the difference in mechanical conditions to which the bone cells were subjected. Furthermore, in supported transplants devitalized parietal bones could form a barrier for diffusion of nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural killer cell activity in patients with ovarian tumors and uterine myomas.

Natural killer (NK) cells are considered to play a role in surveillance against neoplasia. Therefore, the aim of our study was to evaluate peripheral blood NK cell activity in patients with benign and malignant ovarian tumors as well as uterine myomas. NK cell activity was evaluated by means of 51/Cr-release cytotoxicity assay using K562 erythroleukemic cells as a target. We found that natural cytotoxicity of peripheral blood mononuclear cells was significantly (P > 0.01) decreased in patients suffering from both benign and malignant ovarian tumors as compared to healthy women. A degree of NK cell activity abrogation was higher in patients with malignant tumors. Similar decrease was also observed in patients with uterine myomas. Since ovarian tumors and uterine myomas are known to be associated with an increased serum levels of estrogens it is possible that abrogation of NK cell activity in these patients is related to immunosuppression mediated by these hormones.

Production of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by a rat corneal epithelial cell line.

Production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by rat corneal epithelial cells in response to lipopolysaccharide and phorbol-12-myristate-13-acetate (PMA) was tested. Supernatants from rat corneal epithelial cells treated with lipopolysaccharide and PMA were collected after 6, 24 and 48 h and tested with enzyme-linked immunosorbent assay for IL-1 beta, IL-6 and TNF-alpha. The activity of TNF-alpha was additionally confirmed with bioassay on L929 cells. It was found that control groups did not produce significant levels of either cytokine. However, after stimulation with lipopolysaccharide, cells produced mainly IL-6, whereas after PMA they produced mainly TNF-alpha. IL-6 levels 24 and 48 h after PMA stimulation were also elevated, which could have been caused by the presence of TNF-alpha. Production of IL-1 beta in all groups was very low and remained within the test sensitivity range. These results show that the rat corneal epithelial cell line produces inflammatory cytokines in response to proinflammatory mediators. For this reason, it could be used for measuring the effects of irritants on the cornea.

[Experimental closure of bronchial stump after pneumonectomy with the greater omentum]. / Doswiadczalne zamykanie siecia oskrzela glównego po pneumonektomii.

Authors present their results with experimental closure of bronchial stump after pneumonectomy with pedicled flaps of the greater omentum in sheep. In the experimental group left thoracotomy and laparotomy was performed. The left lung was resected and the bronchial stump was closed with the pedicled flap of the greater omentum. In controls standard left pneumonectomy was performed. After different periods of time (1-12 weeks) the animals were sacrificed, the bronchial stumps were excised and submitted to pathological studies. In none case bronchial fistula occurred. Pathological studies showed that the omentum at the base of the bronchial stump was transformed in the fibrous tissue. The bronchial epithelium begins to cover the omentum in 4 weeks after the operation and this process is finished in 12 weeks after the operation. Pedicled flap of the greater omentum seems to be useful in surgical treatment of bronchial fistulas.

[Studies of natural killer cell cytotoxicity against human chondrocytes in patients with articular arthritic changes complicated by psoriasis and systemic scleroderma]. / Badania nad naturalna cytotoksycznoscia komórkowa przeciw ludzkim chondrocytom u chorych ze zmianami stawowymi w przebiegu luszczycy i twardziny ukladowej.

The natural killer (NK) activity against fetal chondrocytes was studied in patients with psoriasis and systemic sclerosis. It was shown that the cell responsible for the cytotoxic effect is a lymphocyte CD16+, CD2-. In patients with arthritic psoriasis NK activity against chondrocytes was significantly higher than in other psoriasis patients or in healthy controls. Preliminary studies show that the NK activity against chondrocytes is decreased in patients with systemic sclerosis.

[Studies on the production of epidermal cytokines after UVB irradiation in patients with epidermodysplasia verruciformis]. / Badania nad produkcja cytokin naskórkowych pod wplywem UVB u chorych z epidermodysplasia verruciformis.

The production of interleukin-1 (IL-1) and interleukin-1 inhibitor by keratinocytes isolated from the skin of epidermodysplasia verruciformis patients was studied. Keratinocytes from uninvolved skin of patients with most pronounced neoplastic lesions produced large amounts of an IL-1 inhibitor (20-40 kD). Keratinocytes from preneoplastic lesions showed no significant differences compared to cells from healthy donors but their production of IL-1 after UVB irradiation was increased.

[Studies on conditions for the culture of rabbit corneal epithelium]. / Badania nad ustaleniem optymalnych warunków hodowli nablonka rogówki królika.

PURPOSE: Research was aimed at comparison of isolation methods as well as determination of growth and differentiation dynamics of rabbit corneal epithelium (CE) in vitro. Adhesion, growth and differentiation of CE cells growing on collagen membranes were evaluated. MATERIAL AND METHODS: Research was performed on the cells of rabbit corneal epithelium isolated mechanically or enzymatically (Dispase II) from comeas excised at the edge of limbus. CE cells were cultured in media with high or low contents of calcium, with addition of FCS, insulin, cholera toxin and EGF. RESULTS: In comparison with mechanical isolation, enzymatic isolation yielded 4-5 times more living undifferentiated (CE) cells. The highest dynamics of in vitro growth was observed in primary cultures in low-calcium medium supplemented with the above substances. After 20 population doublings cells were differentiated and died. Only few cells on collagen membranes adhered to the collagen but did not enter division. CONCLUSIONS: Current research allowed for determination of methodology for CE excision and isolation. Optimal conditions for in vitro growth have been established. Growth dynamics and proliferation of CE in vitro have been evaluated. Growth of CE on standard collagen membrane has not been observed.