Kenneth Ka Ho Lee

Neurotransmitters, Neuropeptides and Calcium Binding Proteins in Developing Human Cerebellum: A Review

Many endogenous neurochemicals that are known to have important functions in the mature central nervous system have also been found in the developing human cerebellum. Cholinergic neurons, as revealed by immunoreactivities towards choline acetyltransferase or acetylcholinesterase, appear early at 23 weeks of gestation in the cerebellar cortex and deep nuclei. Immunoreactivities gradually increase until the first postnatal month. Enkephalin is localized in the developing cerebellum, initially in the fibers of the cortex and deep nuclei at 16–20 weeks and then also in the Purkinje cells, granule cells, basket cells and Golgi cells at 23 weeks onward. Another neuropeptide, substance P, is localized mainly in the fibers of the dentate nucleus from 9 to 24 weeks but substance P immunoreactivity declines thereafter. GABA, an inhibitory neurotransmitter of the central nervous system, starts to appear at 16 weeks in the Purkinje cells, stellate cells, basket cells, mossy fibers and neurons of deep nuclei. GABA expression is gradually upregulated toward term forming networks of GABA-positive fibers and neurons. Catecholaminergic fibers and neurons are also detected in the cortex and deep nuclei at as early as 16 weeks. Calcium binding proteins, calbindin D28K and parvalbumin, make their first appearance in the cortex and deep nuclei at 14 weeks and then their expression decreases toward term, while calretinin appears later at 21 weeks but its expression increases with fetal age. The above findings suggest that many neurotransmitters, neuropeptides and calcium binding proteins (1) appear early during development of the cerebellum; (2) have specific temporal and spatial expression patterns; (3) may have functions other than those found in the mature neural systems; and (4) may be able to interact with each other during early development.

Systemic lupus erythematosus with neuropsychiatric manifestation incurs high disease costs: a cost-of-illness study in Hong Kong

OBJECTIVE To determine the direct and indirect costs of SLE in Hong Kong, and to ascertain the relationship between neuropsychiatric SLE (NPSLE) and disease costs. METHODS A retrospective, cross-sectional, non-randomized cost-of-illness study was performed in a tertiary rheumatology specialty centre in Hong Kong. Participants completed questionnaires on sociodemographics, employment status and out-of-pocket expenses. Healthcare resources consumption was recorded by chart review. The occurrence of NPSLE since onset of SLE was determined using the 1999 ACR nomenclature and standard definitions. Mann-Whitney U-test was used to compare disease costs between patients with and without NPSLE. Multiple linear regression was used to determine the predictors of the costs. RESULTS Three hundred and six Chinese patients were recruited, with a mean age of 41 years and mean disease duration of 9.6 years. A total of 108 NPSLE events were recorded by 83 patients. The most common manifestations were seizure and cardiovascular disease. The mean annual total costs were USD 13,307 per patient. The direct costs dominated the total costs, and the costs of inpatient care contributed 52% of the direct costs. Patients with NPSLE incurred significantly higher direct and indirect costs compared with those without NPSLE. The number of NPSLE events was an independent explanatory variable associated with both direct and indirect costs. CONCLUSION The economic impact of SLE in Hong Kong is considerable and patients with NPSLE incur higher disease costs compared with those without NPSLE. Improvement in prevention of end-organ damage, especially neuropsychiatric manifestation, may reduce costs of SLE patients.

CD146+ Human Umbilical Cord Perivascular Cells Maintain Stemness under Hypoxia and as a Cell Source for Skeletal Regeneration

The human umbilical cord perivascular cells (HUCPVCs) have been considered as an alternative source of mesenchymal progenitors for cell based regenerative medicine. However, the biological properties of these cells remain to be well characterized. In the present study, HUCPVCs were isolated and sorted by CD146+ pericyte marker. The purified CD146+ HUCPVCs were induced to differentiate efficiently into osteoblast, chondrocyte and adipocyte lineages in vitro. Six weeks following subcutaneous transplantation of CD146+ HUCPVCs-Gelfoam-alginate 3D complexes in severe combined immunodeficiency (SCID) mice, newly formed bone matrix with embedded osteocytes of donor origin was observed. The functional engraftment of CD146+ HUCPVCs in the new bone regenerates was further confirmed in a critical-sized bone defect model in SCID mice. Hypoxic conditions suppressed osteogenic differentiation while increased cell proliferation and colony-forming efficiency of CD146+ HUCPVCs as compared to that under normoxic conditions. Re-oxygenation restored the multi-differentiation potential of the CD146+ HUCPVCs. Western blot analysis revealed an upregulation of HIF-1α, HIF-2α, and OCT-4 protein expression in CD146+ HUCPVCs under hypoxia, while there was no remarkable change in SOX2 and NANOG expression. The gene expression profiles of stem cell transcription factors between cells treated by normoxia and hypoxic conditions were compared by PCR array analysis. Intriguingly, PPAR-γ was dramatically downregulated (20-fold) in mRNA expression under hypoxia, and was revealed to possess a putative binding site in the Hif-2α gene promoter region. Chromatin immunoprecipitation assays confirmed the binding of PPAR-γ protein to the Hif-2α promoter and the binding was suppressed by hypoxia treatment. Luciferase reporter assay showed that the Hif-2α promoter activity was suppressed by PPAR expression. Thus, PPAR-γ may involve in the regulation of HIF-2α for stemness maintenance and promoting the expansion of CD146+ HUCPVCs in response to hypoxia. CD146+ HUCPVCs may serve as a potential autologous cell source for bone regeneration.

A New Oxidative Stress Model, 2,2-Azobis(2-Amidinopropane) Dihydrochloride Induces Cardiovascular Damages in Chicken Embryo

It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD50 value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.

Relationship Between Flare and Health-related Quality of Life in Patients with Systemic Lupus Erythematosus

Objective. To investigate (1) the relationship between flares and health-related quality of life (HRQOL) in Chinese patients with systemic lupus erythematosus (SLE) in Hong Kong; and (2) the influence of severity of flare, number of organs involved in flares, and manifestations of flares on HRQOL. Methods. A retrospective study was performed on 303 patients with SLE. Participants completed the Medical Outcomes Survey Short-Form 36 (SF-36) and underwent clinical and laboratory examination to evaluate disease activity and damage. The total number and manifestations of flares during the preceding year were assessed retrospectively. Multiple linear regression analysis was used to identify the independent variables associated with impairment of HRQOL. Results. Patients with flares were younger, had a shorter disease duration, and had higher disease activity at the time of the assessment. A total of 72 episodes of flares were recorded in 61 patients in the preceding year. Patients with flares had significantly lower scores in the areas of role limitation due to physical problems, general health, social function, and role limitation due to emotional problems compared with those without flare. The physical health summary scale was also lower in patients with flares. In the multivariate analysis, the presence of musculoskeletal flare was independently associated with all scales of the SF-36, except bodily pain and mental health. Conclusion. The low level of patients’ HRQOL is mostly associated with the presence of musculoskeletal involvement.

Development of formaldehyde-free agar/gelatin microcapsules containing berberine HCl and gallic acid and their topical and oral applications

The safety issues of biomedical applications have been a major concern in recent years. Drug delivery associated with microencapsulation technology has been focused on as microencapsulated drugs are believed to promote comparative therapeutic efficiency on human absorption and prolong the life cycle of drugs. The most commonly applied crosslinker is formaldehyde in a gelatin microencapsulation system, which is considerably toxic to the human body. To reduce the risks involved when using formaldehyde, agar was associated with gelatin as the wall matrix materials of microcapsules as it could crosslink with gelatin to give a gel network in the microcapsules formation. Here we report the development, characterization and safe use of agar–gelatin microcapsules. We further demonstrate that both oral and topical applications are possible using the berberine HCl and gallic acid loaded microcapsules respectively. Microcapsules containing both drugs were prepared combining the optimal parameters identified. The mean drug loading efficiency and the mean particle sizes of the berberine HCl loaded microcapsules were 78.16% and 16.75 μm respectively, while those of gallic acid loaded microcapsules were 70.28% and 21.98 μm respectively. The compositions and surface morphology of berberine HCl and gallic acid containing microcapsules were examined using Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The in vitro controlled release models demonstrated that the drugs could be gradually released from the microcapsules. The minimum inhibitory concentrations (MICs) and anti-Staphylococcus aureus activity also proved that the berberine HCl loaded microcapsules exhibited better antibacterial activity towards Staphylococcus aureus when compared with those of the original drugs. The in vitro drug delivery model also demonstrated the delivery of berberine HCl from microcapsule treated textiles into nude mice skin. The in vivo mice disease model also showed that gallic acid loaded microcapsules were helpful in the treatment of acute liver and kidney toxicity after an overdose administration of acetaminophen. The development of agar–gelatin microcapsules was demonstrated to be an efficient, deliverable tool for both oral and topical applications.

Cohesion promotes nucleolar structure and function.

Mutations in the cohesin acetyltransferase Eco1 or the cohesin ring compromise nucleolar function in budding yeast. A mutation in Eco1 that is associated with the human disease Roberts syndrome compromises looping interactions at the ribosomal DNA and transcription. Depletion of cohesion in a single cell cycle disrupts nucleolar integrity.

Excess ROS induced by AAPH causes myocardial hypertrophy in the developing chick embryo

BACKGROUND The developing embryo is very sensitive to oxidative stress and excess reactive oxygen species (ROS) generation is often associated with cardiovascular malformation. However, little is known about the adverse effects of ROS during heart morphogenesis, especially during the formation of the atria and ventricles. METHODS AND RESULTS We have treated early chick embryos with 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) to generate free radicals in the developing heart. We established that excess ROS induced by AAPH caused cardiomegaly to develop in 4-, 14- and 17-day-old embryos. The cardiomyocytes of these AAPH-treated hearts were hypertrophic, in both the compact and trabeculated myocardium. The weight of these hearts was also significantly increased in an AAPH dose-dependent fashion. We examined and compared the functions of the AAPH-treated and untreated hearts by echocardiography and determined that the ejection fraction was shortened. BrdU incorporation assay was performed and revealed that cell proliferation was not the main cause of cardiomegaly. However, we established that the cardiomyocytes exposed to excess ROS were distinctively larger than control cardiomyocytes - indicting that cardiomegaly was attributed to hypertrophy. We have also found that excess ROS inhibited Wnt signaling but enhanced VEGF signaling. Consequently, this promoted angiogenesis and caused larger coronary arteries to develop in the AAPH-treated hearts. CONCLUSIONS We have demonstrated that cardiomyocyte hypertrophy and changes in Wnt and VEGF signaling were the main contributing factors in the development of cardiomegaly induced by oxidative stress.

Preparation of 8-hydroxyquinoline derivatives as potential antibiotics against Staphylococcus aureus.

This work describes the preparation of quinoline compounds as possible anti-bacterial agents. The synthesized quinoline derivatives show anti-bacterial activity towards Staphylococcus aureus. It is interesting to observe that the synthetic 5,7-dibromo-2-methylquinolin-8-ol (4) shows a similar minimum inhibitory concentration of 6.25μg/mL as compared to that of methicillin (3.125μg/mL) against Staphylococcus aureus.

Development of hydrocortisone succinic acid/and 5-fluorouracil/chitosan microcapsules for oral and topical drug deliveries

Recently, we demonstrated the safety use of calendula oil/chitosan microcapsules as a carrier for both oral and topical deliveries. We also reported the improved biological activity towards skin cells and Staphylococcus aureus of phyllanthin containing chitosan microcapsules. However, the possibility of both oral and topical applications was still necessary to be further studied. Here we investigated that both oral and topical applications of chitosan-based microcapsules were tested using hydrocortisone succinic acid (HSA) and 5-fluorouracil (5-FU), respectively. The drug loading efficiency, particle size, surface morphology and chemical compositions of both drug loaded microcapsules were confirmed by UV-vis spectrophotometer, particle size analyzer, scanning electron microscope and Fourier transform infrared spectroscopy. The in vitro release studies revealed that both HSA and 5-FU could be released form chitosan microcapsules. The mean adrenocorticotropic hormone concentration in HSA loaded microcapsule mice plasma was detected to be lower than that of water control. One hundred micrograms per milliliter of 5-FU containing microcapsules exhibited a stronger growth inhibition towards skin keratinocytes than that of free 5-FU. In vitro drug delivery model demonstrated the delivery of 5-FU from microcapsule treated textiles into nude mice skin. Further uses of the drug loaded microcapsules may provide an efficiency deliverable tool for both oral and topical applications.