Endogenous tissue engineering: PTH therapy for skeletal repair

Based on its proven anabolic effects on bone in osteoporosis patients, recombinant parathyroid hormone (PTH_1-34) has been evaluated as a potential therapy for skeletal repair. In animals, the effect of PTH_1-34 has been investigated in various skeletal repair models such as fractures, allografting, spinal arthrodesis and distraction osteogenesis. These studies have demonstrated that intermittent PTH_1-34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms, which include effects on mesenchymal stem cells, angiogenesis, chondrogenesis, bone formation and resorption. Furthermore, PTH_1-34 has been shown to enhance bone repair in challenged animal models of aging, inflammatory arthritis and glucocorticoid-induced bone loss. This pre-clinical success has led to off-label clinical use and a number of case reports documenting PTH_1-34 treatment of delayed-unions and non-unions have been published. Although a recently completed phase 2 clinical trial of PTH_1-34 treatment of patients with radius fracture has failed to achieve its primary outcome, largely because of effective healing in the placebo group, several secondary outcomes are statistically significant, highlighting important issues concerning the appropriate patient population for PTH_1-34 therapy in skeletal repair. Here, we review our current knowledge of the effects of PTH_1-34 therapy for bone healing, enumerate several critical unresolved issues (e.g., appropriate dosing regimen and indications) and discuss the long-term potential of this drug as an adjuvant for endogenous tissue engineering.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Di(2-ethylhexyl)phthalate enhances hepatic phospholipid synthesis in rats

The effects of di(2-ethylhexyl)phthalate, a typical peroxisomal proliferator, on the activities of key enzymes in the glycerophospholipid synthetic pathway and the incorporation of lipid precursors into liver lipids in vitro were studied periodically in rats. When di(2-ethylhexyl)phthalate was fed at the 1% level to rats, glycerol-3-phosphate acyltransferase activity increased 2-3-fold in liver homogenates and microsomes in 2-4 days. The specific activity of microsomal CTP:phosphocholine cytidylyltransferase increased by 1.5-fold, whereas the cytosolic activity was depressed. The microsomal CDPcholine:diacylglycerol cholinephosphotransferase specific activity decreased, whereas the activity in the homogenates increased, suggesting the proliferation of the hepatic endoplasmic reticulum in di(2-ethylhexyl)phthalate-treated rats. The incorporation of [1(3)-3H]glycerol or [1-14C]acetate into liver phospholipids in vitro increased in 2 days and stayed at a high level up to 12 days. The present study confirmed that di(2-ethylhexyl)phthalate induced an enhancement of phospholipid synthesis in the liver. The increase in hepatic phospholipid synthesis by this drug is presumably linked to the proliferation of peroxisomes and other intracellular membranes.     

Optical birefringence of phosphatidylcholine liposomes in gel phases

A simple and rapid method for studying optical anisotropic properties of liposomes was proposed. Intensities of transmitted light through one spherical liposome of dipalmitoylphosphatidylcholine placed between two polarizers were measured at various wavelengths by a microscopic spectrophotometer. Large increases in the intensities were observed at the prephase-transition temperature, which were caused by an increase in the birefringence of the multilayer of the liposome. The birefringence values obtained from the intensity data were about 0.020 below the pretransition temperature and 0.028 above that temperature. These values are in good agreement with the results reported for the plane samples in which lipid bilayers are stacked. The obtained values of the birefringence were far lower than the values estimated from polarizabilities. This lower birefringence is attributed to disordering of the tilt direction in the multilayer. The degree of order of the liposome multilayers calculated from the birefringence increased by 38% at the pretransition. This simple method is applicable to the study of the multilayer structure of liposomes in water.     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

Cell- and tissue-specific monoclonal antibodies in eggs and embryos of the ascidian Halocynthia roretzi

To obtain specific immunological probes for studying molecular mechanisms involved in the early embryonic development of ascidians, we have produced monoclonal antibodies directed against a homogenate of larvae of the ascidian Halocynthia roretzi. Among these, we have screened monoclonal antibodies that specifically recognize cells and/or tissues of the embryo. Characterization of six epidermis-specific monoclonal antibodies (including larval tunic-specific and larval fin-specific), three muscle-specific antibodies, two endoderm-specific antibodies, one notochord-specific antibody and two monoclonal antibodies that specifically recognize trunk-lateral cells suggests that these monoclonal antibodies may be useful as markers for analysing molecular mechanisms involved in specification of these cells. Seven monoclonal antibodies characteristically stain intercellular materials of the developing embryo and may therefore be valid for studying cellular construction of the embryo. Furthermore, monoclonal antibodies that recognize components of follicle cells, perivitelline space and sperm have also been established.     

Splenic outer periarterial lymphoid sheath (PALS): An immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2-positive macrophages in the rat

Summary In an attempt to reveal the role of antigen-laden marginal metallophil (MM) and other macrophages in the intrasplenic immune response of a specific B-cell lineage to a thymus-independent type-2 antigen (Ficoll conjugated with fluorescein isothiocyanate), simultaneous immuno-histological observations of the involved cells were performed in the rat. By newly established methods of double or triple immunostainings, time-kinetics of the following parameters were studied and compared: (1) the antigen, (2) the specific antibody-forming cells (AFC) directed to the fluorescein-isothiocyanate determinant, (3) proliferating cells labeled with 5-bromo-2-deoxyuridine (BrdU), and (4) macrophage subpopulations recognized by monoclonal antibodies (ED2 and ED3). The antigen localized stably not only in the marginal-zone macrophages but also in the MM except around the follicular area. The increase of BrdU-positive cells was observed from day 2 up to day 4 after antigen injection mostly in the periphery of the periarterial lymphoid sheath (outer PALS), which indicated antigen-induced proliferation. As a novel finding, the majority of AFC, both BrdU-positive and -negative, were either closely associated with the antigen-laden MM, or forming cell clusters with ED2-positive macrophages in the outer PALS. In contrast, there were very few AFC in juxtaposition to antigen-free MM in the follicular area or the antigen-laden marginal zone macrophages. The results led to the proposal of a hypothesis that the antigen-laden MM together with ED2-positive macrophages constitute an immunoproliferative microenvironment for the plasmacellular reaction by accumulating the antigen-specific B-cell lineage and promoting these cells to differentiate into the AFC and to proliferate in the outer PALS.Abbreviations AFC specific antibody-forming cells - BrdU 5-bromo-2-deoxyuridine - Fic-F FITC-conjugated Ficoll - FITC fluorescein isothiocyanate - HRP horseradish peroxidase - MM marginal metallophils - MZ marginal zone - PALS periarterial lymphoid sheath - PBS phosphate-buffered saline - TI2 thymus-independent type-2     

Differences in growth of transplantable ascites hepatoma among various lines of Donryu rat

A total of 48 Donryu rats from 8 colonies of 5 lines were inoculated intravenously with 10(7) cells of the ascites hepatoma strain AH 66. All the conventional rats of the lines D-1 and D-2 died between 9 and 15 days after inoculation with a good growth of implanted tumor cells. On the other hand, SPF rats of the lines A-1, B-1, C and E survived for 60 days showing complete rejection of the implanted tumor cells. A 50% of conventionalized ex-SPF rats of the lines A-2 and B-2, which had been once established as SPF colonies, and thereafter had been "re-conventionalized", rejected the tumor cells. The present observations indicate that the microbial conditions of the animal, e.g. whether the animal is SPF or not, might play an important role in the growth of the implanted ascites hepatoma.