Monitoring liver function among patients who initiated anti-tuberculosis drugs in Taiwan, 2000-2011.

OBJECTIVE: To investigate adherence to liver function monitoring as recommended in Taiwan's tuberculosis (TB) diagnosis and treatment guidelines for newly diagnosed TB patients. DESIGN: Retrospective cohort study of the National Health Insurance Research Database (NHIRD), Taiwan, 2000-2011. METHODS: From the NHIRD, we identified 11 397 newly diagnosed TB patients who initiated anti-tuberculosis treatment between 2000 and 2011 and categorised these into three groups: completely, partially and non-adherent. Logistic regression was used to explore potential factors associated with the adherence rate. RESULTS: The completely adherent rate increased from 0.5% in 2000 to 9.2% in 2011, while the non-adherent rate decreased from 17.5% to 1.2%. Compared to the non-adherent group, patients with a history of liver disease (OR 4.36, 95%CI 1.92-9.87) and viral hepatitis (OR 9.39, 95%CI 1.47-60.19), as well as patients whose prescribing physicians were specialists in chest (OR 4.59, 95%CI 1.91-11.05), TB (OR 2.55, 95%CI 1.01-6.40) and infectious diseases (OR 3.93, 95%CI 1.08-14.31), had higher odds of being completely adherent to the guidelines. CONCLUSION: Our findings could serve as an important reference for developing effective strategies to improve adherence to guidelines and prevent patients from developing anti-tuberculosis drug-associated hepatotoxicity.

Quantification of macrovesicular and microvesicular hepatic steatosis in rats using 3.0-T ¹H-magnetic resonance spectroscopy.

BACKGROUND AND OBJECTIVE: Hepatic steatosis (HE), which is common among the general population, is present in donor organs, potentially affecting their graft survival as well as the recovery of the donor. Our goal was to develop an experimentally and clinically reliable, noninvasive method to quantify macrovesicular and microvesicular hepatic steatosis using 3-T (1)H-magnetic resonance spectroscopy (MRS). MATERIALS AND METHODS: Macrovesicular and microvescular steatosis were induced in rats using methylcholine deficiency and choline deficiency diets. A MayoBC10 coil was used for radiofrequency transmission and signal recept. Measurements of hepatic fat content were performed using (1)H spectroscopy on a 3.0-T whole-body GE Signa system. The ratio of the areas under the curve of fat (0.8-1.3 ppm) and water (4.7 ppm) was used to determine hepatic fat content, which was compared with the degree of histopathologic and biochemical steatosis. RESULTS: Twenty rats were divided into two groups based on the percentage of microvesicular liver steatosis. Group A (n = 13) was the lower percentage group (microvesicular < 10%) while group B (n = 7), the higher group (microvesicular ≥ 10%). The mean total fatty change in the liver was 58.4% ± 47.2% and 67.6% ± 39.1% in groups A and B, respectively. A highly significant linear correlation between (1)H-MRS and total fatty change was observed in group A (r = .986, P < .001) while there was a relatively poor correlation in group B (r = .764, P = .05). The power to predict fatty change in the liver in groups A and B was significantly different (P = .004). CONCLUSIONS: The degree of hepatic steatosis with a small amount of microvesicular steatosis (<10%) can be precisely predicted using 3-T (1)H-MRS.

Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion.

OBJECTIVE: The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. DESIGN: Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. RESULTS: The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. CONCLUSIONS: (1) Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. (2) The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. (3) Perfusion improved the cell and matrix distribution in the bone region and augmented the integration at the bone-cartilage interface. (4) Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone).

Effects of initial seeding density and fluid perfusion rate on formation of tissue-engineered bone.

We describe a novel bioreactor system for tissue engineering of bone that enables cultivation of up to six tissue constructs simultaneously, with direct perfusion and imaging capability. The bioreactor was used to investigate the relative effects of initial seeding density and medium perfusion rate on the growth and osteogenic differentiation patterns of bone marrow-derived human mesenchymal stem cells (hMSCs) cultured on three-dimensional scaffolds. Fully decellularized bovine trabecular bone was used as a scaffold because it provided suitable "biomimetic" topography, biochemical composition, and mechanical properties for osteogenic differentiation of hMSCs. Trabecular bone plugs were completely denuded of cellular material using a serial treatment with hypotonic buffers and detergents, seeded with hMSCs, and cultured for 5 weeks. Increasing seeding density from 30 x 10(6) cells/mL to 60 x 10(6) cells/mL did not measurably influence the characteristics of tissue-engineered bone, in contrast to an increase in the perfusion rate from 100 microms(-1) to 400 microms(-1), which radically improved final cell numbers, cell distributions throughout the constructs, and the amounts of bone proteins and minerals. Taken together, these findings suggest that the rate of medium perfusion during cultivation has a significant effect on the characteristics of engineered bone.

Roles of microtubules, cell polarity and adhesion in electric-field-mediated motility of 3T3 fibroblasts.

Direct-current electric fields mediate motility (galvanotaxis) of many cell types. In 3T3 fibroblasts, electric fields increased the proportion, speed and cathodal directionality of motile cells. Analogous to fibroblasts' spontaneous migration, we initially hypothesized that reorientation of microtubule components modulates galvanotaxis. However, cells with intact microtubules did not reorient them in the field and cells without microtubules still migrated, albeit slowly, thus disproving the hypothesis. We next proposed that, in monolayers wounded and placed in an electric field, reorientation of microtubule organizing centers and stable, detyrosinated microtubules towards the wound edge is necessary and/or sufficient for migration. This hypothesis was negated because field exposure mediated migration of unoriented, cathode-facing cells and curtailed migration of oriented, anode-facing cells. This led us to propose that ablating microtubule detyrosination would not affect galvanotaxis. Surprisingly, preventing microtubule detyrosination increased motility speed, suggesting that detyrosination inhibits galvanotaxis. Microtubules might enhance adhesion/de-adhesion remodeling during galvanotaxis; thus, electric fields might more effectively mediate motility of cells poorly or dynamically attached to substrata. Consistent with this hypothesis, incompletely spread cells migrated more rapidly than fully spread cells. Also, overexpression of PAK4, a Cdc42-activated kinase that decreases adhesion, enhanced galvanotaxis speed, whereas its lack decreased speed. Thus, electric fields mediate fibroblast migration via participation of microtubules and adhesive components, but their participation differs from that during spontaneous motility.

Disparate aggrecan gene expression in chondrocytes subjected to hypotonic and hypertonic loading in 2D and 3D culture.

The effects of hypotonic (180 mOsm) and hypertonic (580 mOsm) medium loading on chondrocyte aggrecan gene expression in 2D monolayer and 3D hydrogel culture (agarose or alginate) were studied. Aggrecan promoter activity was monitored using a luciferase reporter gene assay and transient transfection. Osmotic loading was observed to differentially affect promoter activity, with hypotonic loading generally producing at least a 40% elevation in promoter activity, except for the case of alginate where a 50% suppression was observed. Hypertonic loading produced at least a 35% decrease in activity for all cultures. Similar osmolality-induced changes to aggrecan mRNA levels were observed in monolayer cells using qPCR. Deletion of exon 1 blocked the sensitivity of monolayer cells to hypertonic but not hypotonic medium changes. Confocal microscopy measurements suggested that the degree of hypotonic swelling in cells encapsulated in 3D matrix was restricted compared to monolayer cells whereas the degree of hypertonic shrinking was similar under both culture conditions.

Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).

Chondrocyte translocation response to direct current electric fields.

Using a custom galvanotaxis chamber and time-lapse digital video microscopy, we report the novel observation that cultured chondrocytes exhibit cathodal migration when subjected to applied direct current (DC) electric fields as low as 0.8 V/cm. The response was dose-dependent for field strengths greater than 4 V/cm. Cell migration appeared to be an active process with extension of cytoplasmic processes in the direction of movement. In some cells, field application for greater than an hour induced elongation of initially round cells accompanied by perpendicular alignment of the long axis with respect to the applied field. Antagonists of the inositol phospholipid pathway, U-73122 and neomycin, were able to inhibit cathodal migration. Cell migration toward the cathode did not require the presence of serum during field application. However, the directed velocity was nearly threefold greater in studies performed with serum. Studies performed at physiologic temperatures (approximately 37 degrees C) revealed a twofold enhancement in migration speed compared to similar studies at room temperature (approximately 25 degrees C). Findings from the present study may help to elucidate basic mechanisms that mediate chondrocyte migration and substrate attachment. Since chondrocyte migration has been implicated in cartilage healing, the ability to direct chondrocyte movement has the potential to impact strategies for addressing cartilage healing/repair and for development of cartilage substitutes.

Social support and life adjustment for teenagers with cleft lip and palate in Taiwan.

BACKGROUND: The teenage years (13-19 years) are a critical period for psychosocial development. Therefore, social support and life adjustment for teenagers with cleft lip/palate were chosen for investigation. METHODS: The sample survey included 101 cleft patients randomly selected from the Chang Gung Craniofacial Center, and 101 non-cleft controls selected from local middle and high schools for comparison. Questionnaires were used to evaluate the social support and life adjustment scales. Items of social support were subdivided into social activities, social service, and psychological support. Items of life adjustment were subdivided into social, psychological, and physical adjustments. RESULTS: The coefficients of alpha reliability were high at 0.9294 for social support, and 0.9389 for life adjustment. The results show that personal factors, family factors, and treatment status do not influence social support or life adjustment in either group; however, gender does. In the control group, non-cleft males had better social support and life adjustment than did females. Such a difference was not observed in the cleft group. Both groups received the same social support. The cleft teenagers have a significantly lower level of life adjustment. The association between the social support and life adjustment is high in both groups. CONCLUSION: The cleft group has a lower level of life adjustment. There is a positive relationship between social support and life adjustment. More social support is required for the cleft group in order to improve their level of life adjustment.

Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization.

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.

[Seroepidemiology of Toxoplasma gondii infection among dogs in Taipei, Taiwan].

Seroprevalence of toxoplasmosis in dogs was investigated in Taipei from July 1995 to June 1996 by the latex agglutination assay. A total of 289 serum samples were taken from stray and domestic dogs as well as from dogs in randomly selected animal hospitals and pet stores. The overall seroprevalence among all dogs was 24.6% (71/289). With regard to the source of the dogs, the seropositivity was 15.4% (10/65) for dogs in animal hospitals, 16.3% (7/43) for dogs in pet stores, 15.5% (11/71) for domestic dogs and 39.1% (43/110) for stray dogs. The seropositivity of stray dogs was significantly higher than that of the domestic dogs (P < 0.05). However, there was no significant difference among the randomly selected places or dog strains (P > 0.05).

[Seroepidemiology of Toxoplasma gondii infection among Atayal aborigines and local animals in Nan-ao district, Ilan county and Jen-ai district, Nan-tou county, Taiwan].

In this study, latex agglutination test (LAT) was used to detect sera anti-toxoplasma antibodies of Atayal aborigines and local animals in Nan-ao district, Ilan county and Jen-ai district, Nan-tou county as a measure of exposure to the Toxoplasma gondii. Out of 422 Atayal aborigines and 64 different animals were tested in Nan-ao district and 82 Atayal children in Jen-ai district were also screened, the positive rates for sera anti-toxoplasma antibodies were 21.8%, 17.2% and 15.9%, respectively. In Nan-ao district, neither were the positive rates significantly different in males (22.1%) and females (21.4%), nor in humans (21.8%) and dogs (19.6%) (P > 0.05). The seroprevalence in adults (28.3%) was significantly higher than that in children (18.7%) (P < 0.05), and the highest seropositive rate (38.1%) was in the age group 50-59 years and the lowest (7.7%) was in the age group 1-9 years. In general, the age pattern of prevalence is consistent with increasing duration of exposure to Toxoplasma gondii with age. For animals, the seropositive rate in dogs (19.6%) was also significantly higher than that in wild rats (7.7%) (P < 0.05). No significant difference in seropositive rate of Atayal children was observed between Nan-ao and Jen-ai districts (P > 0.05).

Seroprevalence of Toxoplasma gondii antibodies among Atayal aboriginal people and their hunting dogs in northeastern Taiwan.

Atayal aborigines, living at an altitude of 1,500-1,600 m in northeastern Taiwan, still hunt for wild animals with the help of hunting dogs. In this study, the latex agglutination test (LAT) was used to detect sera anti-toxoplasma antibodies in this community as a measure of their exposure to Toxoplasma gondii. The positive rates for sera anti-toxoplasma antibodies were 21.8% and 19.6%, respectively, in 422 Atayal and 51 hunting dogs tested. Neither of the positive rates were found to be significantly different between male (22.1%) and female Atayal (21.4%), or between humans (21.8%) and dogs (19.6%) (P > 0.05) when compared by the Chi-Squared test (chi 2-test). A significant difference was observed between the positive rates in adults (28.3%) and children (18.7%) (P < 0.05), and the age pattern of prevalence is consistent with an increasing duration of exposure to Toxoplasma gondii with age. The consumption of raw liver of wild animals or insufficiently cooked meat may be the major mode of transmission of toxoplasmosis in Atayal.

15-Lipoxygenase in human platelets.

The metabolism of arachidonic acid by washed human platelets was investigated. [1-14C]Arachidonic acid was extensively converted to [1-14C]12-hydroxyeicosatetraenoic acid. In addition, several minor labeled products were formed with a considerably lower specific activity, indicating their preferential formation from endogenous substrate. These were dihydroxy metabolites of arachidonic acid with conjugated triene structures and were identified as 14,15-dihydroxyeicosatetraenoic acid (three isomers) and 8,15-dihydroxyeicosatetraenoic acid (three isomers). The identification was based on comparison with reference compounds with respect to chromatographic properties, characteristic UV spectra, and mass spectrometry of several derivatives. Bradykinin (10(-8)-10(-5) M) was found to enhance the formation of all these compounds. In addition, the monohydroxy acid fraction was found to contain 15-hydroxyeicosatetraenoic acid. The present investigation thus demonstrates the occurrence of a 15-lipoxygenase in human platelets in addition to the previously known 12-lipoxygenase.

Deficiency in 15-hydroxyprostaglandin dehydrogenase activity after unilateral ureteral obstruction of the dog kidney.

In the present study, metabolism of prostaglandins (PGs) by 15-hydroxyprostaglandin dehydrogenase (15-OH PGDH) was investigated in dog kidneys with ureteral obstruction. After 24 hr of ureteral obstruction, the obstructed kidney and the contralateral kidney were removed and the cytosolic fractions (105,000 X g), enriched in 15-OH PGDH, were prepared from the cortex and medulla. 15-OH PGDH activity was estimated by radiometric assays of the metabolism of [3H]PGE2 and [3H]prostacyclin. Cortical 15-OH PGDH activity decreased by greater than 50% in the ureter-obstructed kidney as compared to the contralateral kidney. Similar results were obtained by estimating the stereo-specific release of tritium from position 15 using 15-[3H]PGF2 alpha as substrate. In contrast to the cortex, there were no differences in 15-OH PGDH activity found in the medulla of the obstructed and contralateral kidneys. Because the cortex contains higher levels of 15-OH PGDH activity, the deficiency in that site may contribute to the elevated levels of PGs in renal venous blood during ureteral obstruction.

Omega-hydroxylation of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid in human polymorphonuclear leukocytes.

Incubation of arachidonic acid or 12-Ls-hydroxy-5,8,10,14-eicosatetraenoic acid with suspensions of human polymorphonuclear leukocytes led to the formation of 12-Ls,20-dihydroxy-5,8,10,14-eicosatetraenoic acid. The structure of the new metabolite was by physical methods and by chemical degradation.

Nafazatrom (Bay g-6575), an antithrombotic and antimetastatic agent, inhibits 15-hydroxyprostaglandin dehydrogenase.

Nafazatrom (Bay g-6575) is a potent antithrombotic agent which has been suggested to stimulate prostacyclin (PGI2) release from the vascular wall. This study demonstrates that nafazatrom inhibits 15-hydroxyprostaglandin dehydrogenase (15-OH PGDH) of bovine lung and vascular wall. As 15-OH PGDH, the key prostaglandin (PG) catabolizing enzyme, inactivates PGI2 as well as PGE2 and PGF2 alpha, inhibition of this enzyme can result in increased levels of PGI2. Nafazatrom, in the micromolar range, inhibits the metabolism of PGs by 15-OH PGDH in a dose-dependent manner. The IC50 for inhibition of 15-OH PGDH was estimated to be 18.5 microM when [3H]PGF2 alpha was used as substrate. We also estimated nafazatrom-induced changes in PGI2 degradation by lung 15-OH PGDH using the more stable methyl ester (ME) of PGI2 (PGI2-ME) which resists spontaneous degradation. Nafazatrom also inhibited catabolism by 15-OH PGDH of [3H]PGI2-ME to [3H]6,15-diketo-PGF1 alpha ME. These results suggest that the inhibitory action of nafazatrom on 15-OH PGDH contributes to its antithrombotic effect by prolonging the biological half-life of PGI2.

Metabolism of prostacyclin by 9-hydroxyprostaglandin dehydrogenase in human platelets. Formation of a potent inhibitor of platelet aggregation and enzyme purification.

We have reported the identification of a metabolite of prostacyclin (PGI2) in the liver, 6-keto-PGE1, a substance having similar potency to PGI2 in its vascular and antiaggregative actions but differing in its greater stability. Either PGI2 or its inactive hydrolysis product, 6-keto-PGF1 alpha, can be enzymically transformed via the 9-hydroxyprostaglandin dehydrogenase pathway to 6-keto-PGE1. In this study, we demonstrated 9-OH prostaglandin dehydrogenase activity in the cytoplasmic fraction of human platelets by measuring the release of 3H from positin 9 using [9-3H]PGI2 and [9-3H]6-keto-PGF1 alpha as substrates. The enzyme was further purified by DEAE-cellulose, followed by Sephadex G-200, and finally by isoelectric focusing. The enzyme was found to have a pH optimum of 8.5 to 9.0 and an isoelectric point of 5.0. The molecular weight was estimated to be 60,000 by sodium dodecyl sulfate-gel electrophoresis. Enzymic activity was time- and concentration-dependent and required NAD+ as a cofactor. The activity of the purified enzyme was further confirmed by using a more stable form of PGI2, the methyl ester. Incubation of [11-3H]PGI2 methyl ester with the purified enzyme resulted in formation of [11-3H]6-keto-PGE1 methyl ester, which also inhibited platelet aggregation. Thus, 9-hydroxyprostaglandin dehydrogenase in platelets could be a major enzymic pathway for the transformation of PGI2, and perhaps 6-keto-PGF1 alpha, to 6-keto-PGE1. The possibility that the effects of prostacyclin on platelet aggregation are related to conversion to the biologically active metabolite, 6-keto-PGE1, should be considered.