Engineering of implantable cartilaginous structures from bone marrow-derived mesenchymal stem cells.

Fabrication of implantable cartilaginous structures that could be secured in the joint defect could provide an alternative therapeutic approach to prosthetic joint replacement. Herein we explored the possibility of using biodegradable hydrogels in combination with a polyglycolic acid (PGA) scaffold to provide an environment propitious to mesenchymal stem cells (MSCs) chondrogenic differentiation. We examined the influence of type I collagen gel and alginate combined with PGA meshes on the extracellular matrix composition of tissue-engineered transplants. MSCs were isolated from young rabbits, expanded in monolayers, suspended in each hydrogel, and loaded on PGA scaffolds. All constructs (n=48) were cultured in serum-free medium containing transforming growth factor beta-1, under dynamic conditions in specially designed bioreactors for 3-6 weeks. All cell-polymer constructs had a white, shiny aspect, and retained their initial size and shape over the culture period. Their thickness increased substantially over time, and no shrinkage was observed. All specimens developed a hyalin-like extracellular matrix containing glycosaminoglycans (GAGs) and type II collagen, but significant differences were observed among the three different groups. In PGA/MSCs and collagen-PGA/MSCs constructs, the cell growth phase and the chondrogenic differentiation phase of MSCs occurred during the first 3 weeks. In alginate-PGA/MSCs constructs, cells remained round in the hydrogel and cartilage extracellular matrix deposition was delayed. However, at 6 weeks, alginate-PGA/MSCs constructs exhibited higher contents of GAGs and lower contents of type I collagen. These results suggest that the implied time for the transplantation of in vitro engineered constructs depends, among other factors, on the nature of the scaffold envisioned. In this study, we demonstrated that the use of a composite hydrogel-PGA scaffold supported the in vitro growth of implantable cartilaginous structures cultured in a bioreactor system.

[Aortic valve replacement for calcified aortic valve and annulus with ultrasonic decalcification].

UNLABELLED: We conducted ultrasonic decalcification on calcified annulus in patients with aortic stenosis (AS) using an ultrasonic operator, Sonopet (UST 2001) prior to aortic valve replacement (AVR). We studied the reliability of this method. SUBJECT AND METHOD: From January 2002 to August 2005, AVR was conducted for AS using the Sonopet in 45 patients, comprising of 18 male and 27 female subjects. The mean age was 73.3 +/- 9.7. RESULT: Artificial valves were successfully inserted at the intra-annular level in 37 patients and at the supra-annular level in 8 patients without conducting annular enlargement. In the patients with narrow annuli of less than 19 mm (23 patients), the preoperative mean annular diameter was 18.2 +/- 1.0 mm, but significantly larger artificial valves with an average diameter of 19.3 +/- 1.5 mm (p=0.003) were successfully inserted. CONCLUSION: AVR was proved to be safe and easy by previous ultrasonic decalcification of the annuls using the Sonopet. This method was very useful because it required no enlargement of aortic annulus.

Frequency of early and late-onset dementias in a Japanese memory disorders clinic.

The aim of this study was to compare the diagnostic profiles of patients with early (age<65 years) and late (age>or=65 years) onset of dementia in a memory disorders clinic in Japan. A total of 512 consecutive memory clinic patients were evaluated using clinical information and results of examinations. Diagnosis of dementia was made according to DSM-III-R, and that of subtypes according to standard diagnostic criteria. A total of 464 patients met the criteria for dementia. Amongst late-onset patients (n=430), Alzheimer's disease (AD) (48.1%) was the most frequent cause of dementia, followed by AD with cerebrovascular disease (CVD) (31.4%), vascular dementia (VaD) (9.1%), dementia with Lewy bodies (DLB) (3.7%), frontotemporal lobar degeneration (FTLD) (1.6%), and others (5.8%). On the contrary, amongst early onset patients (n=34), the most common dementia diagnosis was AD (38.2%), followed by VaD (23.5%), FTLD (14.7%), AD with CVD (5.9%), DLB (2.9%), and others (17.6%). FTLD and VaD were significantly more common in the early onset group. All patients, but one, with DLB and Parkinson's disease dementia were late-onset. The relative frequencies of AD, VaD, and DLB in our series are consistent with epidemiologic findings in several Western countries; however, the frequency of FTLD is not consistent with the previous findings presenting high frequency in late-onset patients in some Western countries.

Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig's root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Lead content of brain tissue in diffuse neurofibrillary tangles with calcification (DNTC): the possibility of lead neurotoxicity.

Diffuse neurofibrillary tangles with calcification (DNTC) is a form of presenile dementia, characterized pathologically by fronto-temporal atrophy with neurofibrillary tangles (NFTs), neuropil threads and Fahr-type calcification, in which no senile plaques are observed. As already noted, chronic exposure to lead (Pb) might be one of the etiological factors of Fahr-type calcification. Until now, there have been no reports in which Pb concentration has been quantified in DNTC brains. We examined the concentration of Pb in fresh-frozen brain tissue and in 10% formalin-fixed brain tissue from six cases of DNTC, four cases of Alzheimer's disease, and in nine non-demented elderly controls by flameless atomic absorption spectrometry, and demonstrated a high concentration of Pb in DNTC brains. Although it remains unclear how these findings are related to the formation of NFTs, they suggest that Pb neurotoxicity may be involved in the pathogenesis of DNTC.

[Successful treatment of hairy cell leukemia with pentostatin].

A 45-year-old woman was admitted to our hospital in August, 1999. Laboratory data showed a white blood cell count of 5,050/microliter with 78% abnormal lymphocytes, hemoglobin 6.8 g/dl, platelets 4.8 x 10(4)/microliter, and soluble IL-2 receptor 97,600/ml. The abnormal cells were characterized by a hairy appearance under phase contrast microscopy, and showed strong tartrate-resistant acid phosphatase activity. Immunophenotype analysis revealed that these cells were positive for CD11c, CD19 and CD25, and negative for CD5. Bone marrow biopsy showed diffuse proliferation of hairy cells with moderate myelofibrosis. We diagnosed the patient as having European-American-type hairy cell leukemia. Pentostatin was administered at a dose of 5 mg/m2 weekly. After twelve doses, the peripheral blood data returned to the normal range with no hairy cells in the blood or bone marrow, although slight splenomegaly remained. The patient underwent splenectomy in December of the same year, and we were unable to find any hairy cells by histological and immunohistochemical examination. Although most patients with hairy cell leukemia in Japan have the Japanese variant, and the European-American type is rare, pentostatin is as effective as it is for European and American patients.

Caspase activation in high-pressure--induced apoptosis of murine erythroleukemia cells.

Murine erythroleukemia cells were subjected to physicochemical stresses such as high pressure (60--110 MPa), heating (42--45 degrees C), and ultraviolet (UV) irradiation (5--25 kJ/cm(2)). After exposure to these stresses, the cells were cultured at 37 degrees C and atmospheric pressure. The number of the cells treated at 100 MPa, 45 degrees C, or 20 kJ/cm(2) remained constant or decreased at an early stage of culture. The nuclear morphology, agarose gel electrophoresis, and flow cytometry of these cells showed that they undergo apoptosis. The activity of caspase-3 was observed in cells subjected to each stress. In particular, caspase-3 was most readily activated by high pressure under our conditions. The caspase-3 activity and apoptosis exhibited a similar pressure dependence. It is important that both caspase-3 activation and apoptosis induced by high pressure were significantly suppressed by a caspase-3 inhibitor. These results suggest that high-pressure-induced apoptosis is also characterized by the activation of caspase-3, as seen with heat- and UV-induced apoptosis.

Replacing factor-dependency with that for lysozyme: affordable culture of IL-6-dependent hybridoma by transfecting artificial cell surface receptor.

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for industrial applications such as protein production is often not practical. Based on our previous design of a binary hen egg lysozyme (HEL)-specific receptor composed of portions of the anti-HEL antibody and the erythropoietin receptor, a new pair of chimeric receptors having the intracellular domain of gp130 were made and transfected to an interleukin-6 (IL-6)-dependent hybridoma, 7TD1. The clone expressing the two new receptors showed clear HEL dose-dependent cell growth and monoclonal antibody production in both serum-based and serum-free media without IL-6. These results establish the feasibility of applying receptor design to tailor cells for the inexpensive induction of cell growth for the purpose of producing therapeutic products.

Diffuse neurofibrillary tangles with calcification (a form of dementia): X-ray spectrometric evidence of lead accumulation in calcified regions.

Diffuse neurofibrillary tangles with calcification (DNTC) is a form of slowly progressive dementia in which no senile plaques are observed. The calcification is one of the most characteristic features of DNTC. We examined the elemental content of certain mineral deposits (lead, magnesium, phosphorus, calcium, iron, copper and zinc) in the calcified and non-calcified regions of eight cases of DNTC, five cases of Alzheimer's disease (AD) and in eight non-demented elderly controls. The study was performed using a combination of scanning electron microscopy and X-ray spectrometry on 10% formalin-fixed brain tissue. A marked abundance of calcium and phosphorus was observed in the calcified regions of DNTC and non-DNTC brains. Although no lead was observed in the non-calcified regions of DNTC and in non-DNTC brains, traces of lead were detected exclusively in the calcified regions of DNTC brains. The implications and possible significance of the lead accumulation in DNTC brains are discussed.

Plaque-like structures and arteriosclerotic changes in "diffuse neurofibrillary tangles with calcification" (DNTC).

"Diffuse neurofibrillary tangles with calcification" (DNTC) is a rare form of slowly progressive dementia characterized by temporal or fronto-temporal atrophy with neuronal loss and astrocytosis, neurofibrillary tangles and Fahr-type calcification, but no senile plaques in the cerebral cortex. In patients with DNTC, we detected a novel histopathological abnormality that we termed "plaque-like structures" (PLS). PLS appeared as oval, slightly eosinophilic masses of up to 100 microns in diameter. With methenamine silver stain, the PLS were argyrophilic, and thread-like structures were observed in and around them. Most PLS were observed in deep layers of the cortex and subcortical white matter, and were accompanied by small vessels. They were intimately associated with the small-vessel walls and astrocytes. They were composed of two types of fibers. The first type comprised straight and loosely interwoven fibers about 25-30 nm in diameter, while the other type evoked tangles. These structures have not been found in other neurodegenerative diseases, including Alzheimer's disease. In addition, to evaluate hyaline arteriosclerosis in DNTC, we examined sclerotic changes of the medullary arteries and assessed white matter lesions in affected patients. In three of four patients with DNTC, sclerosis of the medullary arteries was significantly more extensive than in age-matched controls. In all four patients, the severity of white matter lesions was graded as moderate or severe in the temporal lobe and as mild or moderate in the frontal lobe. Arteriosclerotic changes and white matter lesions can occur without hypertension and beta amyloid deposits in DNTC.

Purification and characterization of a basic amino acid-specific peptidase from seeds of jack bean (Canavalia ensiformis).

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degrees C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.

Moving on to the cargo problem of microtubule-dependent motors in neurons.

Vigorous investigation has finally begun to shed light on the cargo problem of the microtubule-dependent motors, kinesin and dynein superfamily proteins. Biochemical observations have suggested that the potential cargoes of certain populations of motor proteins seem to be in vesicle-form, each vesicle possessing specific functional marker molecules. In addition to the close relationship between microtubule-dependent motors and cargoes in vesicle-form, kinesin has also been highlighted as an apparent driving force for another cargo in non-vesicle-form, cytoplasmic protein. On the basis of new biophysical and cell-biological evidence, the controversy over the movement of cytoplasmic cargoes has entered a new phase.

High pressure sensitizes murine erythroleukemia cells to caffeine-induced premature mitosis.

Murine erythroleukemia (MEL) cells were exposed to a high pressure of 80 MPa or aphidicolin (APH), DNA polymerase inhibitor. The effects of caffeine on cell cycle were examined using these cells. During the culture of 80 MPa-treated MEL cells at atmospheric pressure, the cells arrested in the G2 phase, and cyclin B and hyperphosphorylated p34(cdc2) were accumulated. Namely, maturation promoting factor (MPF) composed of p34(cdc2) and cyclin B was inactive. However, upon exposure to caffeine, these cells entered prematurely into mitosis by activating MPF. Caffeine-induced premature mitosis was suppressed by butyrolactone I and orthovanadate. On the other hand, APH-treated MEL cells, which were not exposed to 80 MPa, were not so sensitive to caffeine-induced premature mitosis despite cyclin B accumulation. In this case, dephosphorylation of p34(cdc2) was not induced by caffeine. Interestingly, caffeine-induced premature mitosis in the 80 MPa-treated cells was also suppressed by APH. These results suggest that the premature mitosis of 80 MPa-treated MEL cells by caffeine is induced by active MPF, and that APH-sensitive molecules such as DNA polymerase may also play an important role in the checkpoint that controls the transition from G2 to M phase.

Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons.

Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser scanning microscopy and fluorescence correlation spectroscopy. Here, from the pharmacological experiments and the functional blocking of kinesin motor protein by anti-kinesin antibody, we show that the directional movement of fluorescent profile was dependent on kinesin motor function. The fluorescent correlation function and estimated translational diffusion time revealed that tubulin molecule was transported in a unique form of large transporting complex distinct from those of stable polymers or other cytosolic protein.

[Aspergillosis with non-cavitary lung cancer].

Growth of Aspergillus was observed in the necrotic tissue of non-cavitary lung cancer. Case 1 was a 60-year-old male complaining of cough and fever and was found to have left lower lobe atelectasis on chest X-ray. Bronchoscopic examination showed the yellow soft lesion occluding the left upper and lower lobe bronchus. Specimen from the lesion demonstrated adenocarcinoma and hyphae of Aspergillus in the necrotic tumor. Case 2 was a 53-year-old male who was found to have a left upper lobe mass by chest X-ray screening. Bronchoscopic examination showed the yellow polypoid lesion occluding the left upper division bronchus. Pneumonectomy was done and resected specimen of the lesion demonstrated adenocarcinoma and the hyphae of Aspergillus growing in both necrotic tissue and cancer tissue. As both patients had hyphae of Aspergillus in the necrotic tumor, it is considered that the presence of necrosis may cause the growth of Aspergillus.

Primary structure and autoproteolysis of brevilysin H6 from the venom of Gloydius halys brevicaudus.

The complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. H6 has an amino-terminal pyroglutamic acid and consists of a total of 419 residues. An N-linked sugar chain is attached at Asn-181. The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu(98)-Leu(99) or Asp(207)-Ile(208) bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis. Calcium ions also contributed to the stability of H6 against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.

Tau pathology in diffuse neurofibrillary tangles with calcification (DNTC): biochemical and immunohistochemical investigation.

Diffuse neurofibrillary tangles with calcification (DNTC) represents a primary and sporadic presenile dementia that is characterized by temporal or fronto-temporal atrophy with diffuse neurofibrillary tangles (NFTs), neuropil threads and Fahr-type calcification without senile plaques. We examined the tau pathology in five autopsy cases of DNTC by immunoblotting and immunohistochemistry using phosphorylation-dependent and -independent anti-tau antibodies. The pattern of staining for different epitopes of beta-amyloid (A beta) was also investigated. NFTs were immunopositive with all the anti-tau antibodies used in this study. On the immunoblots, sarkosyl-insoluble tau appeared as three major bands of 60, 64 and 68 kDa, and as a minor band at 72 kDa. The majority of extracellular NFTs were weakly immunopositive only with the antibody recognizing the 40 carboxyl-terminal of A beta in DNTC. These results suggest that Alzheimer's disease-like tau pathology could exist independently of A beta deposits in DNTC.

Inducing proliferation of human amniotic epithelial (HAE) cells for cell therapy.

Probably because amnion is derived from the fetus and is exposed to the maternal immune system, human amniotic epithelial (HAE) cells do not express the HLA-A, -B, -C, or -DR antigens on their surfaces, suggesting that HAE cells do not induce rejection (immune reaction) after allotransplantation. And the amnion, like the placenta, is useless to the mother and child after birth. Therefore, HAE cells or tissues were expected to be suitable for allotransplantation. Because HAE cells produce large amounts of enzymes, amnion transplantation has been carried out in order to correct inborn errors of metabolism by supplementing lysosomal enzyme deficiencies. However, several problems remain before amnion allotransplantation can be accepted as effective. The HAE cell population is limited, because the maximum number of HAE cells obtainable from one donor is about 2 x 10(8) cells, and HAE cells proliferate poorly in in vitro culture. In this study, we aimed at increasing the HAE cell population in vitro. First, we investigated the effect of several cytokines on HAE cell proliferation and found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), and transforming growth factor-beta stimulated it, whereas IL-6 and LIF inhibited it. Second, we investigated the effects of amniotic fluid on HAE cell proliferation and observed that IL-6 in amniotic fluid inhibits it. Then, to inhibit the dying of cells, we attempted to inhibit apoptosis (one mode of cell death). Treatment with caspase III inhibitor increased the cell viability of HAE cells by 20%.

Tissue engineering in the twenty-first century.

In the 20th century, free tissue transfers have been successfully introduced using microvascular anastomosis techniques. Transplants not only include whole organs such as the kidney, liver and lung, but also bone, muscle and skin. However, there are a limited number of organs available for transplantation. This leads to the patient not only suffering from the malfunctioning tissue or organ, but also from the psychological trauma of an indefinite waiting period. The rapidly evolving field of tissue engineering is beginning to have an impact on free tissue transfers including organ. Small biopsy specimens can be grown into a large number of cells. These cultured cells can then be seeded onto biodegradable polymers, which serve several purposes. Firstly, the polymers function as a cell delivery system that enables the transplantation of a large numbers of cells into an organism. Secondly, they create a three-dimensional space for cell growth and serve as a template, thereby providing a structure for the extracellular matrix. These approaches have been demonstrated as practical strategies for the reconstruction of many tissues such as the liver, intestines, heart valve leaflets, bone and cartilage.

Ussuristatin 2, a novel KGD-bearing disintegrin from Agkistrodon ussuriensis venom.

Two platelet aggregation inhibitors, ussuristatin 1 (US-1) and 2 (US-2), were newly isolated from the venom of Chinese viper (Agkistrodon ussuriensis) by SP-Toyopearl 650M column chromatography and reverse-phase HPLC. The Mrs of these polypeptides were estimated to be about 8,000 by SDS-PAGE. Analytical gel filtration revealed that US-2 exists as a dimer. Both polypeptides comprised 71 amino acids, whose sequences showed high similarities to those of other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-Gly-Asp (KGD). US-1 strongly suppressed platelet aggregation induced by ADP, collagen, thrombin, and epinephrine with IC50 = 17-33 nM. US-2 also inhibited the platelet aggregation, but the IC50s were about ten times higher. US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin, while US-2 did not inhibit the cell adhesion to fibronectin. This indicates that the KGD-bearing disintegrin is a specific inhibitor for the fibrinogen receptor.