Cuticle network and orientation preference of photonic crystals in the scales of the weevil Lamprocyphus augustus.

This paper reports the structural and optical investigations of the structural colour of the weevil Lamprocyphusaugustus The photonic crystal structure within the weevil's scales was investigated using sequential focused ion-beam milling and scanning electron microscopy imaging. We carefully analysed the reconstructed three-dimensional structure to determine the unit cell of the photonic crystal. It was found that the cuticle network of the cubic unit cell perfectly matches the previously reported diamond-based network. However, different results were obtained for the crystal orientations of the small crystal domains that comprise the entire photonic crystal structure in the scales: <111> directions are highly preferred along the surface normal of the scale. This finding explains the fact that the scale is almost uniformly coloured despite the multi-domain structure. It is confirmed experimentally and theoretically that the wavelength range of the reflection band corresponds to the gap of the photonic band.

Identification of cell-type-specific mutations in nodal T-cell lymphomas.

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations.

Simultaneous treatment of PVC and oyster-shell wastes by mechanochemical means.

This work presents a new process for dechlorinating poly-vinyl chloride (PVC) by the use of oyster-shell waste. The process consists of milling of PVC waste with oyster-shell waste, followed by washing the milled sample with water. The milling of PVC and oyster-shell mixture results in size reduction and rupture in bonds, leading to mechanically induced reactions between the two to form CaCl2 and hydrocarbon with C=C bonds. Washing the milled mixtures with water at room temperature allows complete removal of chlorine from the milled sample. More than 95% of chlorine in PVC was removed when 2h grinding is conducted for the mixture. The present process could offer a potential route to the handling and disposal of oyster-shell and PVC wastes.

Isolation and cultivation of porcine hepatocytes for extracorporeal artificial liver support system.

OBJECTIVE: To develop procedures for the successful harvesting of large quantities of viable and functional pig liver cells from abattoir organs. METHODS: The procedure included partial liver lobe retrograde perfusion and mechanical/enzymatic digestion of the liver tissue, followed by separation of the hepatocytes, based on size and density, from contaminating cell types. RESULTS: Digestion of the partial liver lobe resulted in an average yield of 1.39 x 10(9) cells (9.9 x 10(8) cells/g liver) with an average viability of 92.5%. The yield and viability of cells were improved by dispase/collagenase resultant digestion. The emergence of blebby cells was blocked by supplying oxygen to the cell isolation buffers. Isolated hepatocytes seeded onto polystyrene surfaces remained viable and functional at a level comparable to that of rat hepatocytes, although their function decreased over time. CONCLUSIONS: Adult pig hepatocytes can be harvested with high yields and retain viability and differentiated function using this method. Abattoir pig livers can be an excellent source of hepatocytes for use as the biological component of artificial liver assist devices.

The in vitro differentiating capacity of nonparenchymal epithelial cells derived from adult porcine livers.

Specific nonparenchymal epithelial cell (NPEC) clusters derived from normal adult porcine livers demonstrate a characteristic developmental pattern in the presence of other types of nonparenchymal cells in vitro. This pattern includes scattering, colonial growth, and an emergence of duct-like structures (DLSs) in the colonies. It has been confirmed that 96% of the scattered cell clusters in these cultures develop into colonies containing DLSs. In this study, we examine the differentiation of NPEC clusters using the scattered formation as a marker of the DLS-emerged colonies. We report that the NPECs expressed albumin, alpha-fetoprotein, transferrin, cytokeratin (CK) 18, CK7, and c-met, but not alpha-1-antitrypsin (AAT), at the scattering stage. In addition, at the same stage, NPECs expressed oval-cell-related markers such as OV6, but not biliary epithelial cell (BEC) markers such as gamma-glutamyltransferase, CK19, and CK14. At the DLS emerging stage, hepatocyte markers, including AAT, were detectable in the cells either at the periphery of colonies or in the cells surrounded by the DLSs. On the other hand, the cells constituting DLSs expressed BEC markers, suggesting a bile duct nature of the DLSs. Furthermore, the cells in the colonies possessed an ultrastructural appearance of differentiated hepatocytes and BECs. These results suggest that certain NPECs are bipotent, and that, in culture, they mimic hepatoblast development in vivo.

Sensitization following Thymoglobulin and Atgam rejection therapy as determined with a rapid enzyme-linked immunosorbent assay. US Thymoglobulin Multi-Center Study Group.

Monoclonal and polyclonal anti-thymocyte preparations play an important role in solid organ transplant immunosuppression. While it is generally accepted that blocking anti-idiotypic antibodies can decrease the efficacy of retreatment with mouse monoclonal antibody preparations, sensitization levels and subsequent effects on treatment efficacy are less clear for polyclonal preparations. Serum samples were obtained from 148 patients participating in a multicentre, double-blind randomized phase III trial comparing Atgam (Pharmacia Upjohn, horse anti-thymocyte globulin) with Thymoglobulin (SangStat Medical Corporation, rabbit anti-thymocyte globulin). Recipients of a first or second renal allograft undergoing biopsy proven acute rejection were randomized to treatment with Atgam or Thymoglobulin. Serum samples were analysed for presence of anti-thymoglobulin and anti-Atgam antibodies. Sensitization levels to rabbit IgG in Thymoglobulin-treated patients (68%, n = 54) was similar to sensitization to horse IgG in Atgam-treated patients (78%, n = 54) (two-sided p value = 0.4, Fisher's exact test), although Atgam-treated patients remained sensitized longer (at day 90, 67% anti-horse IgG positive in Atgam treated vs 24% anti-rabbit IgG in Thymoglobulin positive, p = 0.001). No difference was seen in the production of a crossreactive response. Similarly, sensitization had no significant effect on treatment success or failure. For Thymoglobulin-treated patients, the sensitization rate in successfully treated patients was 68%, while inpatients with treatment failures it was 71% (p = not significant, ns). In Atgam-treated patients, the sensitization rate in successfully treated patients was 82%, while in patients with treatment failures it was 67% (p = ns). In conclusion, patients treated with Thymoglobulin and patients treated with Atgam exhibited similar levels of sensitization, presensitization and crossreactive sensitization, although the anti-horse response was longer lasting; neither presensitization nor treatment-induced sensitization appeared to effect treatment efficacy.

A randomized, double-blinded comparison of Thymoglobulin versus Atgam for induction immunosuppressive therapy in adult renal transplant recipients.

BACKGROUND: The aim of this study was to compare the efficacy and safety of Thymoglobulin (a rabbit-derived polyclonal antibody) to Atgam (a horse-derived polyclonal antibody) for induction in adult renal transplant recipients. METHODS: Transplant recipients (n=72) were randomized 2:1 in a double-blinded fashion to receive Thymoglobulin (n=48) at 1.5 mg/kg intravenously or Atgam (n=24) at 15 mg/kg intravenously, intraoperatively, then daily for at least 6 days. Recipients were observed for at least 1 year of follow-up. RESULTS: By 1 year after transplantation, 4% of Thymoglobulin-treated patients experienced acute rejection compared with 25% of Atgam-treated patients (P=0.014). The rate of acute rejection was lower with Thymoglobulin than Atgam (relative risk=0.09; P=0.009). Rejection was less severe with Thymoglobulin than Atgam (P=0.02). No recurrent rejection occurred with Thymoglobulin compared with 33% with Atgam (P=NS). Patient survival was not different, but the composite end point of freedom from death, graft loss, or rejection, the "event-free survival," was superior with Thymoglobulin (94%) compared with Atgam (63%; P=0.0005). Fewer adverse events occurred with Thymoglobulin (P=0.013). Leukopenia was more common with Thymoglobulin than Atgam (56% vs. 4%; P<0.0001) during induction. The mean absolute lymphocyte count remained below baseline with Thymoglobulin throughout the study (P<0.007), but with Atgam, significant lymphocyte reductions occurred only at day 7. The incidence of cytomegalovirus disease was less with Thymoglobulin than Atgam at 6 months (10% vs. 33%; P=0.025). CONCLUSIONS: Brief (7-day) induction with Thymoglobulin resulted in less frequent and less severe rejection, a better event-free survival, less cytomegalovirus disease, fewer serious adverse events, but more frequent early leukopenia than induction with Atgam. These results may in fact be explained by a more profound and durable beneficial lymphopenia.

Colonial growth and differentiation of epithelial cells derived from abattoir adult porcine livers.

The parenchymal cell fraction was isolated from abattoir adult porcine livers and cultured in Dulbecco's Modified Eagles' medium/Ham's F12 medium (DMEM/F12; 1:1) medium supplemented with 5% foetal calf serum, 10 ng/mL glucagon, 10 microg/mL insulin, 60 ng/mL hydrocortisone and eight other factors (NAIR-1 medium). The fraction contained a number of epithelial cells other than hepatocytes, some of which attached to the culture plates as cell clusters and began to grow after 3 days in culture. These epithelial cells growing as colonies were found to express cytokeratin 18 by immunocytochemistry. After 7-8 days, duct-like structures emerged in the central parts of the colonies. The cells constituting the duct-like structures and some cells located outside the structures were positive for cytokeratin 19 and gamma-glutamyltransferase (GGT). The albumin-positive cells were located in the outer parts of the colonies rather than their central parts. Albumin was also detectable in the cells surrounded by the duct-like structures. Moreover, cytochrome P450 IA1 was induced by 3-methylcholanthrene (3-MC) on day 16. These results suggest that porcine liver epithelial cell clusters may contain stem-like cells which can differentiate into mature hepatocytes or bile duct epithelial cells.

Carboxyl-terminal parathyroid hormone fragments stimulate type-1 procollagen and insulin-like growth factor-binding protein-5 mRNA expression in osteoblastic UMR-106 cells.

The effects of carboxyl-terminal (C-) PTH fragments, (35-84), (53-84) and (69-84), on the proliferation and function of osteoblastic UMR-106 cells were compared with those of amino-terminal (N-) (1-34) and intact (I-) (1-84) PTH. I-PTH as well as N-PTH at 10(-8)M significantly inhibited [3H] thymidine incorporation and stimulated alkaline phosphatase activity in UMR-106 cells. No C-PTH fragments affected them. In contrast, the expression of type-1 procollagen mRNA in these cells was stimulated by all C-PTH fragments, inhibited by N-PTH and not affected by I-PTH. All C-PTH fragments except (69-84) as well as N-PTH and I-PTH stimulated IGFBP-5 mRNA expression. The present study suggests that the C-portion of the PTH molecule exercises biological activities in mRNA expression of type-1 procollagen as well as IGFBP-5 in osteoblasts, and that it might be involved in the anabolic action of PTH on bone in vivo.

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface.

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03-3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver.

Effects of 22-oxacalcitriol on bone metabolism in vitro: comparison with calcitriol--effects of 22-oxacalcitriol on osteoclast-like cell formation and bone-resorbing activity.

22-Oxacalcitriol (OCT), a synthetic vitamin D3 analog, can mimic the ability of calcitriol to differentiate leukemia and skin cells, to enhance the immune response and to suppress parathyroid hormone secretion, but has much less calcemic activity than that of calcitriol. The mechanism of this selective action remains not fully understood, and the actions of OCT on bone metabolism are little known. The present study was, therefore, designed to investigate the effects of OCT and calcitriol on: the proliferation and functions of osteoblastic MC3T3-E1 cells; osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells as well as from unfractionated bone cells in the presence of stromal cells; bone resorption; and the proliferation of MC3T3-E1 cells via monocytes. 22-Oxacalcitriol and calcitriol inhibited [3H]thymidine (TdR) incorporation, alkaline phosphatase activity and collagen synthesis of MC3T3-E1 cells to a similar degree. Both OCT (10(-10)-10(-8) mol/l) and calcitriol significantly and similarly stimulated osteoclast-like cell formation from both hemopoietic blast cells and unfractionated bone cells. 22-Oxacalcitriol (10(-10) and 10(-8) mol/l) significantly stimulated bone resorption, although to a slightly lesser degree than did calcitriol. Human monocyte-conditioned medium (CM) significantly stimulated TdR incorporation into MC3T3-E1 cells. On the other hand, CM obtained from monocytes treated with calcitriol (10(-10)-10(-8) mol/l) significantly inhibited TdR incorporation in a dose-related fashion, whereas CM obtained from monocytes treated with OCT (10(-10)-10(-8) mol/l) significantly stimulated TdR incorporation in a dose-related fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Competition between c-fos and 1,25(OH)2 vitamin D3 in the transcriptional control of type I collagen synthesis in MC3T3-E1 osteoblastic cells.

Interaction between c-fos and 1,25(OH)2 vitamin D3 (VD) on the type I collagen synthesis was studied. VD inhibited collagen synthesis and type I collagen mRNA expression in MC3T3-E1 osteoblastic cells. In contrast, VD reversed the inhibition of collagen synthesis and mRNA expression of the c-fos transfectants that overexpressed c-fos gene to a comparable level as those of the control transfectants. The gel shift assay showed the vitamin D receptor (VDR) complex binding to vitamin D responsive element (VDRE) was inhibited under constitutively expressed c-fos gene, suggesting that c-fos gene product, c-Fos, may inhibit the binding of VDR complex to VDRE by making a c-Fos-VDR complex. The result suggests the existence of a fine tuning between c-fos and VD in the bone metabolism which may be relevant to the pathogenesis of rheumatoid bone lesion.

Effect of retinoic acid on the proliferation and alkaline phosphatase activity of osteoblastic MC3T3-E1 cells by modulating the release of local regulators from monocytes.

There has been some evidence suggesting an important role of mononuclear cells at bone remodeling sites in the coupling of bone formation to bone resorption. Since cells of the monocyte-macrophage lineage produce important local regulators of bone remodeling, we examined effects of human monocytes-conditioned medium (CM) treated with retinoic acid on [3H] thymidine incorporation (TdR) and alkaline phosphatase (ALP) activity of osteoblastic MC3T3-E1 cells. Treatment of MC3T3-E1 cells with retinoic acid (10(-8) to 10(-6) M) caused an inhibition of TdR in a dose-dependent manner and an inhibition of ALP activity at 10(-6) M. Conditioned medium from monocytes untreated with retinoic acid caused a stimulation of TdR and an inhibition of ALP activity in these cells. In contrast, treatment of monocytes with retinoic acid (10(-8) or 10(-6) M) abolished both stimulation of DNA synthesis and inhibition of ALP activity induced by CM. The present study suggested that retinoic acid modulated osteoblast proliferation and ALP activity not only directly but also indirectly, presumably through modulating the release of local regulators as to bone remodeling from monocytes.

Second messenger signaling of c-fos gene induction by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic osteosarcoma cells: its role in osteoblast proliferation and osteoclast-like cell formation.

The present study was performed to clarify second messenger signaling in parathyroid hormone (PTH)-induced c-fos gene expression, to characterize the participation of the c-fos gene in the regulation of osteoblast proliferation and function as well as osteoclast-like cell formation by PTH and to compare these effects of PTH with those of PTH-related peptide (PTHrP). Both human (h) PTH-(1-34) and hPTHrP-(1-34) at 10(-8) M induced a transient c-fos gene expression to a similar degree in osteoblastic osteosarcoma cells, UMR-106. N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) as well as Sp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS), an activator of cAMP-dependent protein kinase (PKA), induced a weak c-fos gene expression. Although Rp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), an inhibitor of PKA, almost completely antagonized dbCAMP- and Sp-cAMPS-induced expression of c-fos gene, it did not cause an obvious inhibition of PTH- or PTHrP-induced expression. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induced an intense expression of the c-fos gene, while 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), incapable of activating PKC, and calcium ionophores (A23187 and ionomycin) did not. Protein kinase C inhibitor (H-7, 50 microM) completely blocked the expression of the c-fos gene by PTH as well as by PTHrP). Antisense oligodeoxynucleotides (as-ODN) complementary to c-fos mRNA, which have been shown to inhibit its mRNA translation, at 1 microM significantly antagonized PTH- and PTHrP-induced inhibition of [3H] thymidine incorporation and stimulation of osteoclast-like cell formation in the presence of osteoblasts, but not an increase in alkaline phosphatase activity, compared to control oligodeoxynucleotides with same nucleotides as as-ODN but with a random sequence. The present study indicates the involvement of PKC system in c-fos gene expression by PTH as well as PTHrP and also indicates the involvement of the c-fos gene in the regulation of bone cell physiology by PTH and PTHrP.