Airborne particle dispersion around the feet of surgical staff while walking in and out of a bio-clean operating theatre.

BACKGROUND: Bacterial contamination by airborne particles is one of the most important factors in the pathogenesis of surgical-site infections. AIM: This study aimed to identify the generation and behaviour of airborne particles around the feet of surgical staff while walking in and out of an operating theatre. METHODS: Two physicians and two nurses walked in and out of a bio-clean theatre under laminar airflow, either individually or as a group. The generation and behaviour of airborne particles was filmed using a fine-particle visualization system, and the number of airborne particles per 2.83 m3 of air was counted using a laser particle counter. Each action was repeated five times, and particle counts were evaluated statistically. FINDINGS: Airborne particles were generated from the floor and by the shoes and gown hems of the participants, whether walking individually or as a group. Numerous airborne particles were generated by the group, and significantly more particles, especially those measuring 0.3-0.5 µm, were carried up to the level of the operating table by the group than by individuals (P<0.01). CONCLUSIONS: The results of this study provide a clearer picture of the dispersion and distribution of airborne particles around the feet of staff walking in and out of an operating theatre. The findings suggest that to reduce the incidence of bacterial contamination and risk of surgical site infections, surgical staff should walk calmly and independently, if possible, near sterile areas.

Nanobacteria-like particles in human arthritic synovial fluids.

We investigated the existence of nanosize particles in synovial fluids of rheumatoid arthritis and osteoarthritis patients. These specimens were cultured under mammalian cell culture conditions (37 degrees C; 5% CO2/95% air) for a long period. After about 2 months, many nanoparticles appeared and they gradually increased in number and in size. The nanobacteria-like particles exist in synovial fluids of arthritis patients. The possibility of their existence and pathogenesis in various diseases should be verified cautiously.

A 30-base-pair element in the first intron of SOX9 acts as an enhancer in ATDC5.

SOX9 is a transcription factor that is essential for chondrogenesis and testis differentiation, and haploinsufficiency of SOX9 causes campomelic dysplasia, severe skeletal malformation syndrome with variably penetrant XY sex reversal. Here we demonstrate that in several cell lines that express SOX9, 30-bp element in the first intron of human SOX9 gene act as a potential enhancer in the ATDC5 chondroprogenitor cell line, despite the apparent absence of cell-specific regulatory elements within a 5.5-kb promoter region. Deletion and site-specific mutational analyses reveal that the last 12 bp of the 30-bp element are critical for transcriptional activity, while 5'-half sequences are necessary for full transactivation. Gel retardation assays indicate the possible involvement of several binding factors along the length of this element. These results suggest that functionally interdependent elements in the 30-bp enhancer region of the first intron account for basal expression levels of Sox9 in ATDC5.

Irreversible unfolding of myoglobin in an aqueous solution by supercritical carbon dioxide.

The conformational changes in myoglobin, treated by microbubbling of supercritical carbon dioxide (SC-CO(2)), were investigated by measuring the circular dichroism spectra in the ultraviolet range and compared with those in other proteins (ovoalbumin, bovine serum albumin, and beta-lactoglobulin). Irreversible unfoldings were observed after the microbubbling of SC-CO(2) at 35 degrees C and 30 MPa for 30 min. The degree of unfolding depended on the number of intramolecular S-S bonds. alpha-Helix contents of myoglobin decreased with increasing density of SC-CO(2). Unfoldings of myoglobin induced by heating, pH-lowering, and the addition of a denaturant were reversible. The irreversible unfolding of myoglobin was also observed by the bubbling of gaseous CO(2) under atmospheric pressure, but heating was required.

Dexamethasone inhibition of TGF beta-induced cell growth and type II collagen mRNA expression through ERK-integrated AP-1 activity in cultured rat articular chondrocytes.

Intraarticular injection of dexamethasone (DEX) accelerates cartilage degradation due to the suppression of chondrocyte proliferation and extracellular matrix formation. The present study first demonstrated the interaction between DEX and TGF beta, a potent growth factor for cultured rat articular chondrocytes (CRAC), and then investigated the molecular mechanism by which DEX counteracts TGF beta-induced chondrocyte proliferation and differentiation through the regulation of AP-1 activity. DEX reduced serum-deprived and TGF beta-stimulated cell growth and [(3)H]-thymidine incorporation of CRAC. DEX also inhibited the expression of (alpha)1 type II collagen with concomitant suppression of the promoter activity. Transfection studies using a reporter vector with AP-1 responsive elements showed that DEX reduced TGF beta-activated but not basal luciferase activities. Activation of 3TP-luc, another AP-1 responsive element containing reporter was also blocked by DEX. GAL4-Elk1 studies revealed that DEX suppressed TGF beta-induced ERK activation which led to c-fos gene expression followed by increase in AP-1 complex formation, whereas the Smad pathway was not involved in DEX-dependent negative regulation of AP-1 in a reporter assay that requires FAST1-Smad2 for the activation. DEX also eliminated TGF beta-induced c-fos mRNA expression and ERK activation in Northern analysis and in vitro kinase assay, respectively. Further, DNA synthesis and transactivation of type II collagen by TGF beta were inhibited by PD98059, an inhibitor of MEK. Our results indicate that DEX suppressed TGF beta-induced chondrocyte proliferation and type II collagen expression, probably through selective inhibition of ERK integrated AP-1 activation.

Unfolding of poly-L-glutamic acid by microbubbling of supercritical carbon dioxide.

The conformational changes in alpha-helical poly-L-glutamic acid caused by microbubbling supercritical CO2 were investigated with circular dichroism spectra. After microbubbling using a micropore filter at 35 and 30 MPa for 30 min, alpha-helix content decreased to 37%, while without the filter it was 68%. The alpha-helix structure was significantly decomposed by a high density of CO2. No important changes were observed in heating, autoclaving, or pH-lowering.

Activation of specific MEK-ERK cascade is necessary for TGFbeta signaling and crosstalk with PKA and PKC pathways in cultured rat articular chondrocytes.

OBJECTIVE: TGFbeta is a potent stimulator of cell growth in cultured rat articular chondrocytes (CRAC). The stimulatory effect is mediated through the immediate induction of c-fos gene by activating ERK of MAPK. The present study was undertaken to investigate the upstream regulators involved in TGFbeta-induced ERK activation in CRAC and to compare the results with the events in HepG2 cells. RESULTS: In vitro kinase and trans-reporting assays showed that TGFbeta preferentially activated ERK and JNK pathways in CRAC and HepG2, respectively. ERK activation in CRAC was selectively inhibited by PD98059, a MEK inhibitor. Overexpression of wild or active forms of MEKK1, the upstream activator of ERK and JNK, decreased the TGFbeta-induced 3TP-luciferase activity in CRAC. In contrast, in HepG2 dominant negative form of MEKK1 or SEK1 ligand-dependent reporter activity was diminished. Transfection of TAK1, another MAPKKK, also positively and negatively regulated 3TP transcriptional activity of HepG2 and CRAC, respectively. Activation of PKA by 8-bromo-cyclic AMP or forskolin, and inhibition of PKC by calphostin C, resulted in a significant decrease in 3TP activity as well as in vitro ERK kinase activity in CRAC. CONCLUSIONS: The results indicate that TGFbeta transduces a predominant signal pathway through MEK-ERK-Elk1, independent of MEKK1 or TAK1 pathway in CRAC. However, in HepG2, activation of MEKK1 and TAK1 is essential for TGFbeta-induced signal transmission. The results also demonstrated that in CRAC, MEK-ERK pathway activated by TGFbeta is negatively regulated by PKA cascade but transactivated by PKC.

Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation.

Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-beta1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-beta1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-beta1, we performed in vitro kinase assays using specific substrates. TGF-beta1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240 min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-beta1-induced cell growth by 40%. These results indicate that TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-beta1.

Effect of 22-oxa-1,25-dihydroxyvitamin D3 on human thyroid cancer cell growth.

To examine whether synthetic vitamin D3 analog, 22-oxa-1,25(OH)2D3 (OCT) has an inhibitory effect on the growth of thyroid carcinoma, we tested the in vitro and in vivo effects of OCT on the growth of a well-differentiated thyroid cancer cell line, NPA. OCT bound to its receptor at the same rate as 1,25(OH)2D3, and inhibited the proliferation of NPA cells in vitro in a dose-dependent manner, similar to that observed with 1,25 (OH)2D3. Northern blot analysis showed that steady-state and fetal bovine serum-stimulated levels of c-myc mRNA were suppressed after 0.5-4 hour treatment with OCT. Transfection studies with the deletion mutants of the 5'-up-stream flanking region of c-myc/chloramphenicol acetyltransferase chimera genes indicated the presence of an OCT responsive element between -410 and -106. Next, we examined OCT effects in implanted NPA tumor cells in nude mice. OCT showed no remarkable hypercalcemic effect compared to 1, 25 (OH2)D3, but OCT and 1, 25 (OH2)D3, had no significant inhibitory effect in vivo after either intra-tumor or intra-peritoneum injection. Our results demonstrate that OCT inhibits the proliferation of well-differentiated thyroid cancer in an in vitro system associated with the suppression of c-myc mRNA, but this inhibitory effect was not reproducible in in vivo model.

Regulation of c-fos gene induction and mitogenic effect of transforming growth factor-beta1 in rat articular chondrocyte.

We have previously reported that type I transforming growth factor beta (TGF-beta1) is a potent stimulator of cell growth in articular chondrocytes. In this study, we examined the mechanism of TGF-beta1 induced cellular proliferation by using cultured rat articular chondrocytes (CRAC). A time-course study of [3H]thymidine incorporation upon TGF-beta1 (1 ng/mL) or 10% fetal bovine serum stimulation revealed that TGF-beta1 directly stimulates DNA synthesis in CRAC. Pretreatment with H7, an inhibitor for protein kinase C (PKC), completely blocks TGF-beta1-induced proliferation. Since TGF-beta1 has been shown to transduce signals through MAP kinase cascades, we investigated the induction of several protooncogenes by Northern blotting. TGF-beta1 addition causes an immediate and transient induction of c-fos but not myc or jun mRNA. Furthermore, this c-fos expression is not inhibited by cycloheximide, but is completely abolished by pretreatment with TPA, so that the c-fos gene is a direct target of TGF-beta1 signalling and PKC is involved in this c-fos induction. To refine our understanding of TGF-beta1 regulation of the c-fos promoter region, we performed chloramphenicol acetyltransferase (CAT) assays. A serial deletion analysis of the c-fos promoter region reveals a TGF-beta1 responsive element in a region between -403 and -329 bp upstream of the transcription initiation site. We attempted gel shift assays on this response element with CRAC nuclear extracts. Although this region contains a sis-inducible binding element, we fail to detect specific DNA-protein complexes. Our results, however, suggest that TGF-beta1 acts as a primary mitogen in CRAC and this mitogenic activity requires PKC activation. Finally, the subsequent induction of c-fos expression occurs through an as yet unidentified transactivation mechanism.

cDNA cloning and chromosomal mapping of rat Smad2 and Smad4 and their expression in cultured rat articular chondrocytes.

Smad proteins are known to transduce signalling of TGF-beta receptor superfamily. We report here the entire sequences of rat Smad2 and Smad4 which have not been identified yet. Entire sequences were identified by degenerated polymerase chain reaction and following phage library screening and 5' RACE. The predicted amino acid sequences of rat Smad2 and Smad4 are highly conserved among rat, human and mouse. We also mapped these Smads to chromosome 18q.12.3. Unlike endothelial cells, TGF-beta1 stimulates articular chondrocyte proliferation as well as extracellular matrix production, and acts as a repairing agent against cartilage destruction. Since both Smad2 and Smad4 are essential factors for TGF-beta signalling, we examined their expression and regulation in cultured articular chondrocytes. Northern blot analysis showed that TGF-beta1 significantly increased the mRNA level of Smad2 but not of Smad4 in a dose- and time-dependent manner, suggesting that the augmentation of TGF-beta1 action is caused by increasing the expression of the downstream signalling molecule.

Inactivation of Bacillus spores by the supercritical carbon dioxide micro-bubble method.

Bacillus spores were effectively inactivated by the supercritical (SC) CO2 micro-bubble method. The micro-bubble SC CO2 treatment of B. cereus, B. subtilis, B. megaterium, B. polymyxa, and B. coagulans at 40 degrees C and 30 MPa for 30 min produced greater reduction (about 3 log cycles of reduction) than a similar treatment without a filter. The SC CO2 treatment of B. polymyxa, B. cereus, and B. subtilis spores at 45 degrees C, 50 degrees C, respectively, and 30 MPa for 60 min resulted in a 6-log cycle reduction of survival. The SC CO2 treatment under the foregoing conditions should offer higher efficiency than that of heat treatment at 100 degrees C for 60 min. In addition, the SC CO2 treatment (30 MPa, 60 degrees C, 30 min) of B. polymyxa and B. cereus spores also produced a 6-log cycle reduction.

Expression of parathyroid hormone-related peptide in human osteoarthritis.

To evaluate the involvement of the expression of parathyroid hormone-related peptide gene in human articular cartilage pathology, we performed immunohistochemical staining and in situ hybridization on specimens of femoral head cartilage obtained from 15 patients with osteoarthritis, 11 with rheumatoid arthritis, and 12 control subjects. Parathyroid hormone-related peptide-positive chondrocytes were observed predominantly in degenerated lesions of osteoarthritic tissue and were less evident in rheumatoid arthritic samples, while the normal cartilage expressed little parathyroid hormone-related peptide. In addition, the level of parathyroid hormone-related peptide expression was clearly dependent on the degree of cartilage degeneration; cartilage tissues with moderate degenerative changes contained more positive chondrocytes compared with mildly or severely degenerated cartilage. In situ hybridization confirmed the localization of parathyroid hormone-related peptide protein and demonstrated intense expression of mRNA of the peptide in osteoarthritic samples. This is the first demonstration of parathyroid hormone-related peptide expression in chondrocytes from pathologic articular cartilage of humans. Our results suggest that parathyroid hormone-related peptide may be involved in the pathophysiology of osteoarthritis.

Localisation of apoptosis and expression of apoptosis related proteins in the synovium of patients with rheumatoid arthritis.

OBJECTIVES: To investigate whether apoptosis occurs in the synovium of rheumatoid arthritis (RA), and the intermediate molecules operating in this process. METHODS: DNA fragmentation was detected by in situ nick end labelling (ISNEL) in the synovium of patients with RA (n = 11) and control patients with femoral neck fracture (n = 5). The expression of proteins p53, p21WAFI/CIPI, c-myc, proliferating cell nuclear antigen (PCNA), and Bcl-2 was also examined by immunohistochemistry. RESULTS: ISNEL positive synovial cells with apoptosis specific morphology were detected in extremely limited areas in only two RA synovial tissue specimens. Proteins p53, p21WAFI/CIPI, and c-myc, known inducers of apoptosis or cell cycle arrest or both, were expressed in the sublining cells independent of ISNEL positive cells. PCNA, a marker for cell proliferation, was observed in the synovial lining cells. Bcl-2, an inhibitor of apoptosis, was expressed mainly in infiltrated lymphocytes and in parts of the sublining layer cells of RA; it also did not correspond with ISNEL staining. CONCLUSIONS: Our findings indicate that RA synovial cells undergo apoptosis in addition to cell proliferation, but the frequency of apoptosis was very low. We suspect that the apoptotic process in the RA synovium may be suppressed by over-expression of Bcl-2. Although expressed proteins p53, p21WAFI/CIPI, and c-myc were present in the RA synovium, these protooncogenes are probably not implicated in the apoptotic process.

[Malignant choroid plexus papilloma. An autopsy case report (author's transl)].

Malignant choroid plexus papilloma is a rare disease. The autopsy case of malignant choroid plexus papilloma being suggestive of the youngest in Japan, was reported. This 8-months-old baby had normal delivery history, and the development and growth were not eventful. The patient admitted to the University hospital because of projecting vomiting and meningeal irritating signs at 7.5 months in age, and septic meningitis was most suspected. He died 2 weeks afte the admission. At autopsy, a large papillary tumor with marked necrosis and hemorrhage was seen in the right lateral ventricle. The right lateral ventricle was almost replaced by the tumor. The metastasis to the brain base and the sheeding to the subarachinoidal space of the cerebellum were noted.