Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Controlled release by biodegradable hydrogels enhances the ectopic bone formation of bone morphogenetic protein

The objective of this study is to develop a carrier for the controlled release of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of the bone regeneration activity. Hydrogels with different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Following subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled BMP-2 into the back of mice, the in vivo retention period of BMP-2 prolonged with a decrease in the water content of hydrogels used, although every time period was much longer than that of BMP-2 solution injection. Ectopic bone formation studies demonstrated that the alkaline phosphatase (ALP) activity and osteocalcin content around the implanted site of BMP-2-incorporated gelatin hydrogels were significantly high compared with those around the injected site of BMP-2 solution. The values became maximum for the gelatin hydrogel incorporating BMP-2 with a middle period of BMP-2 retention, while bone formation was histologically observed around the hydrogel incorporating BMP-2. The ALP activity was significantly higher than that of the collagen sponge incorporating BMP-2. We concluded that the controlled release technology of BMP-2 for a certain time period was essential to induce the potential activity for bone formation.     

Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells

PROBLEM: The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. METHOD OF STUDY: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. RESULTS: The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. CONCLUSIONS: These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.     

Preparation of blood-compatible hollow fibers from a polymer alloy composed of polysulfone and 2-methacryloyloxyethyl phosphorylcholine polymer

Blood-compatible hollow fibers were successfully prepared from a polymer alloy composed of polysulfone (PSf) and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer. To improve the hydrophilicity, fouling-resistance characteristics, and blood compatibility of the PSf hollow fiber in a hemodialyzer, an MPC polymer that can be blended with PSf was synthesized in order to prepare the polymer alloy (PSf/MPC polymer). The contents of the MPC polymer blended in the PSf were 7 and 15 wt%. The PSf/MPC polymer hollow fiber could be prepared by both wet and dry-wet processing methods. The hollow fiber took an asymmetric structure, that is, the hollow-fiber membrane had a dense skin layer on the porous sponge-like structure. The mechanical strength was higher than that of conventional PSf hollow fibers for hemodialysis. The surface characterization of the PSf/MPC polymer hollow fiber by x-ray photoelectron spectroscopy revealed that the MPC units were concentrated at the surface. The permeability for solutes through the PSf/MPC polymer hollow fibers was measured for 4 h. The permeabilities of both a low-molecular-weight compound and protein were greater than those of the PSf hollow fibers. The amount of adsorbed protein was lower on the PSf/MPC polymer hollow fiber when compared to that of the PSf hollow fiber. Moreover, platelet adhesion was also effectively inhibited on the PSf/MPC polymer hollow fiber. Based on these results, the addition of the MPC polymer to the PSf is a very useful method to improve the functions and blood compatibility of the hollow fiber.     

Ganglioglial Differentiation in Medulloblastoma

A case of cerebellar medulloblastoma with clusters of mature ganglion cells and glial cells is described. The patient, a 15 -year -old girl, underwent three operations followed each time by radiation and chemotherapy during the four-year clinical course. Histologically, the ganglion cells were clearly identifiable by their abundant eosino-philic cytoplasm, round nuclei with prominent nucleoli, tigroid granules, and argyrophilic fibrils and axons. Im-munohistochemically, the cells were NSE- and NF positive, and ultrastructurally they contained abundant tubules and filaments, neurosecretory granules and well developed rough endoplasmic reticulum. There were many cells transitional in appearance between primitive cells and mature ganglion cells. The tumor also had many mature yet atypical astrocytes and oligodendrocytes. The exact mechanism of the extensive neuronal and glial maturation of medulloblastoma cells is unclear, but the repetitive surgical interventions, radiation and chemotherapy might have had certain cytostatic effects on rapidly dividing medulloblastoma cells, giving them a chance to mature into postmitotic cells with potential for neuronal and glial differentiation. Acta Pathol Jpn 40: 50–56, 1990.     

In vitro and ex vivo blood compatibility study of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated hemodialysis hollow fibers

To identify the advantages of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated polysulfone (PSf) hollow fibers for hemodialyzer and hemofilter minimodules with hollow fibers were made and blood compatibility was evaluated in vitro and ex vivo. Three types of hollow fibers, i.e., pure PSf (no additives), PSf alloyed with poly(1-vinyl-2-pyrrolidone) (PVPy), and PSf coated with the MPC copolymer, were processed in wet conditions. Commercially available hollow fibers (APS) were used as a control sample. The PSf hollow fibers have a condensed structure. A porous structure was observed when the PVPy was alloyed before wet processing, and no effect of the innercoated MPC copolymer on the porous structure was observed. One-tenth-sized minimodules of the conventional hemodialyzer were fabricated with 200 fibers each. The solute permeability of the hollow fibers was evaluated using 10% bovine serum in a buffer solution containing cytochrome C, which is a model protein of ₂-microglobulin. After circulation for 2.5h, the solute permeability of APS and PVPy-alloyed PSf hollow fibers decreased to 50% compared with their initial values. In contrast, the value for the hollow fibers innercoated with the MPC copolymer maintained its initial level. The inner surface of the dialysis membranes was observed with a transmission electron microscope and a layer of adsorbed protein on the PSf, APS, and PVPy-alloyed PSf hollow fibers was observed, but not on the MPC copolymer-coated fibers. Blood cell adhesion was then evaluated by circulation of whole rabbit blood without any anticoagulant ex vivo. Many adherent cells were observed on the PVPy-alloyed PSf hollow fibers; however, blood cells did not adhere or aggregate on the MPC copolymer-coated hollow fibers. From these results, we concluded that the in-situ coating of MPC copolymer on PSf hollow fibers is effective in preventing blood coagulation and maintaining the solute permeability of the fibers.     

Use of collagen sponge incorporating transforming growth factor-beta1 to promote bone repair in skull defects in rabbits.

The objective of this study was to evaluate the potential of collagen sponge incorporating transforming growth factor-beta1 (TGF-beta1) to enhance bone repair. The collagen sponge was prepared by freeze-drying aqueous foamed collagen solution. Thermal cross-linking was performed in a vacuum at 140 degrees C for periods ranging from 1 to 48 h to prepare a number of fine collagen sponges. When collagen sponges incorporating 125I-labeled TGF-beta1 were placed in phosphate-buffered saline (PBS) solution at 37 degrees C, a small amount of TGF-beta1 was released for the first hour, but no further release was observed thereafter, irrespective of the amount of cross-linking time the sponges had received. Collagen sponges incorporating 125I-labeled TGF-beta1 or simply labeled with 125I were implanted into the skin on the backs of mice. The radioactivity of the 125I-labeled TGF-beta1 in the collagen sponges decreased with time; the amount of TGF-beta1 remaining dependent on the cross-linking time. The in vivo retention of TGF-beta1 was longer in those sponges that had been subjected to longer cross-linking times. The in vivo release profile of the TGF-beta1 was matched with the degradation profile of the sponges. Scanning electron microscopic observation revealed no difference in structure among sponges subjected to different cross-linking times. The TGF-beta1 immobilized in the sponges was probably released in vivo as a result of sponge biodegradation because TGF-beta1 release did not occur in in vitro conditions in which sponges did not degrade. We applied collagen sponges incorporating 0.1 microg of TGF-beta1 to skull defects in rabbits in stress-unloaded bone situations. Six weeks later, the skull defects were covered by newly formed bone, in marked contrast to the results obtained with a TGF-beta1 free empty collagen sponge and 0.1 microg of free TGF-beta1. We concluded that the collagen sponges were able to release biologically active TGF-beta1 and were a promising material for bone repair.     

Natural antibodies against the immunoglobulin F(ab′)2 fragment cause elimination of antigens recognized by the F(ab′)2 from the circulation

A humanized monoclonal IgG1 antibody, designated hC4G1, recognizes the fibrinogen receptor glycoprotein (GP)IIb/IIIa on platelets and inhibits platelet aggregation. When the F(ab′)₂ fragment of hC4G1 (F(ab′)₂ hC4G1) was administered to cynomolgus monkeys, all the monkeys showed inhibition of platelet aggregation ex vivo. Unexpectedly, a significant decrease in platelet count was observed in 5 of 18 monkeys. Antibodies against F(ab′)₂ hC4G1 were detected in the plasma of these monkeys by ELISA. Antibody activity in the plasma of these monkeys was significantly correlated with the intensity of platelet decrease (r = 0.84). The natural monkey antibodies to F(ab′)₂ hC4G1 were directed against the C-terminal region of F(ab′)₂ fragment common to all human and humanized IgG antibodies. Natural homo-reactive antibodies were also detected in human plasma from 15 of 40 healthy volunteers. Specificity was closely similar to that of the monkey antibodies. Affinity-purified human homo-reactive antibodies enhanced phagocytosis of platelets treated with the F(ab′)₂ hC4G1. Monkey plasma with high homo-reactive antibody activity was confirmed to decrease platelet count when administered together with F(ab′)₂ hC4G1 to a monkey with low antibody activity. These results suggest that F(ab′)₂ of humanized and human antibodies causes elimination of the corresponding antigens from the circulation by homo-reactive antibodies.     

Juvenile hyaline fibromatosis: A report of two unrelated adult sibling cases and a literature review

Two unrelated adult sibling cases (36- and 32-year-old females) of Juvenile hyaline fibromatosis are presented. The parents of one of these patients were non-consanguineous but natives of a small Island, and one elder sister among four siblings was affected with the same disease. The parents of the other patient were consanguineous, and one other sibling suffered from the identical disease. Both patients presented with multiple subcutaneous nodules, which they had had since infancy, and had undergone numerous surgical excisions. Light microscopy examination of skin lesions from both patients showed identical histology; an abundance of a homogenous, amorphous, eosinophlllc extracellular matrix in which spindle-shaped cells were embedded. Electron microscopically, the spindle-shaped cells had hypertrophic Golgi apparatus and dilated, rough endoplasmlc reticulum. Fine flbrillar and granular material-filled structures, the contents of which were occasionally released into the extracellular matrix, were also seen, immunohistochemically, the spindle-shaped cells were vlmentin-positive but negative for α-smooth muscle actln and S-100 protein, and the hyaline ground substance was positive for type I and type III collagen but negative for type II and type IV collagen and tenascin. Matrix metalloprotelnase-1, -2, and -9, and tissue inhibitor of matrix metalloproteinase (TlMP)-2 was positive but TIMP-1 was negative. A review of 39 cases of juvenile hyaline fibromatosis In the literature is also presented. In summary, skin lesions may be the most outstanding symptoms of juvenile hyaline fibromatosis, but joint contracture and gingival hypertrophy precede the skin manifestation.     

The role of the interleukin-2 (IL-2)/IL-2 receptor pathway in MRL/lpr lymphadenopathy: The expanded CD4−8− T cell subset completely lacks functional IL-2 receptors

Autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice spontaneously develop a systemic lupus erythematosus-like disease accompanied by a profound lymphadenopathy that consists of CD4^?8^?B220⁺ a P T cells. By the use of cross-linking experiments with radiolabeled interleukin-2 (IL-2), these abnormal T cells have been reported to constitutively express the IL-2 receptor β chain (IL-2Rα), a signal transducing component of IL-2R, in the absence of the a chain (IL-2Rα).To critically reevaluate the role of the IL-2/IL-2R pathway in the pathogenesis of lymphadenophathy we examined expression of the IL-2Rα and IL-2Rβ in MRL/lpr mice by ¹²⁵I-IL-2 binding analysis and also by flow cytometric analysis using monoclonal antibodies against each component of the receptor. We found that, contrary to the previous report, the CD4^?8^?B220⁺ α β T cells in lymph node (LN) of MRL/lpr mice were negative for both IL-2Rα and IL-2Rβ expression. The lpr liver CD4^?8^?B220⁺ a P T cells that had been implicated in the genesis of these abnormal LN T cells were also negative for IL-2Rβ expression. Therefore, our results indicate that the IL-2/IL-2R system plays little role, if any, in the expansion of abnormal CD4^?8^? B220⁺ α β T cells in MRL/lpr mice.