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Burns in pregnancy--maternal and fetal prognosis

Cheah SH, Sivanesaratnam V

Aust N Z J Obstet Gynaecol, 1989 May;29(2):143-5.
PMID: 2803125

In this series the incidence of pregnancy in women in the reproductive age group admitted to hospital with burns was 7.8% (9 of 116). The maternal and perinatal outcome is related to the extent, presence or absence of complications of burns and to the gestational age of the fetus. Two maternal deaths in this series occurred in patients with burns involving more than 85% of the skin surface; in both instances stillbirths occurred less than 48 hours after the burns. In view of the high perinatal mortality, patients with extensive burns who are more than 32 weeks' pregnant should be delivered soon after admission. The extensively burned anterior abdominal wall can make assessment of uterine size difficult. An assessment in such a situation would be useful.
Generation and characterization of a high-affinity monoclonal antibody for MUC1 measurement in breast cancer

Hamid SS, Cheah SH

Hybridoma (Larchmt), 2011 Apr;30(2):137-43.
PMID: 21529286 DOI: 10.1089/hyb.2010.0091

Breast mucin is secreted by breast tumor cells and serves as a marker for breast cancer. Thus, antibodies against breast mucin will be valuable in the development of immunotherapy and laboratory diagnostic tests. Monoclonal antibodies (MAbs) against breast cancer-associated antigen were generated and characterized. Balb/c mice were immunized with breast cancer-associated antigen CA15-3, and subsequently splenocytes from immunized mice were fused with myeloma cells. After fusion, culture supernatants from hybridomas surviving HAT medium were screened by enzyme-linked immunosorbent assay (ELISA). A total of eight hybridomas producing MAbs against breast cancer showed significant levels of antibody activity against CA15-3. Two selected stable hybridomas were adapted into CELLine CL 350 bioreactors, and the MAbs produced were characterized for their subclass, specificity, and affinity. The MAbs were of high specificity and affinity as shown by ELISA. The MAbs produced may represent a powerful tool and are considered promising reagents for use in diagnosis and detection of early stage of the disease.
Heterogeneity in cancer cells: variation in drug response in different primary and secondary colorectal cancer cell lines in vitro

Arul M, Roslani AC, Cheah SH

In Vitro Cell Dev Biol Anim, 2017 May;53(5):435-447.
PMID: 28120247 DOI: 10.1007/s11626-016-0126-x

Tumor heterogeneity may give rise to differential responses to chemotherapy drugs. Therefore, unraveling tumor heterogeneity has an implication for biomarker discovery and cancer therapeutics. To test this phenomenon, we investigated the differential responses of three secondary colorectal cancer cell lines of different origins (HCT116, HT29, and SW620 cells) and four novel primary cell lines obtained from different colorectal cancer patients to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) and explored the differences in gene expression among the primary cell lines in response to exposure to cytotoxic drugs. Cells were exposed to different doses of 5-FU and L-OHP separately or in combinations of equitoxic drug or equimolar drug ratios (median effect of Chou-Talalay principle). Cell viability was assessed using MTT assay and the respective IC50values were determined. Changes in gene expression in primary cell lines after exposure to the same drug doses were compared using real-time PCR array. The sensitivities (IC50) of different cell lines, both secondary and primary, to 5-FU and L-OHP were significantly different, whether in monotherapy or combined treatment. Primary cell lines needed higher doses to reach IC50. There were variations in gene expression among the primary cell lines of different chemosensitivities to the challenge of the same combined dose of 5-FU and L-OHP. The results confirm the heterogeneous nature of colorectal cancer cells from different patient tumors. Studies using primary cancer cells established from patient's tumors rather than secondary cell lines will more closely reflect the actual character of the disease.
Humanization of chimeric anti-CD20 antibody by logical and bioinformatics approach with retention of biological activity

Khoo YL, Cheah SH, Chong H

Immunotherapy, 2017 06;9(7):567-577.
PMID: 28595518 DOI: 10.2217/imt-2017-0016

AIM: To develop a fully bioactive humanized antibody from the chimeric rituximab for potential clinical applications using a relatively simpler and faster logical and bioinformatics approach.
METHODS: From bioinformatics data, mismatched mouse amino acids in variable light and heavy chain amphipathic regions were identified and substituted with those common to human antibody framework. Appropriate synthetic DNA sequences inserted into vectors were transfected into HEK293 cells to produce the humanized antibody.
RESULTS: Humanized antibodies showed specific binding to CD20 and greater cytotoxicity to cancer WIL2-NS cell proliferation than rituximab in vitro.
CONCLUSION: A humanized version of rituximab with potential to be developed into a biobetter for treatment of B-cell disorders has been successfully generated using a logical and bioinformatics approach.
Fulltext Cardiovascular reactivity in Malay Moslems during Ramadan

Husain R, Cheah SH, Duncan MT

Singapore Med J, 1996 Aug;37(4):398-401.
PMID: 8993142

The investigation examined the possibility that observance of Ramadan by Moslems in Malaysia is associated with modification of circulatory parameters. Cardiovascular reactivity was investigated employing the cold hand immersion test as the stressor stimulus. Resultant data showed increased blood pressures and vascular resistance during Ramadan in the absence of cold stimulus while the magnitude of the maximal cardiac and vascular response to the applied stressor which served as indicators of reactivity was not affected by the Ramadan situation.
Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells

Chan YY, Kim KH, Cheah SH

J Ethnopharmacol, 2011 Oct 11;137(3):1183-8.
PMID: 21810462 DOI: 10.1016/j.jep.2011.07.050

ETHNOPHARMACOLOGICAL RELEVANCE: Sargassum polycystum, a type of brown seaweed, has been used for the treatment of skin-related disorders in traditional medicine.
AIM OF THE STUDY: The aim of the present study is to investigate the antimelanogenesis effect of Sargassum polycystum extracts by cell-free mushroom tyrosinase assay followed by cell viability assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells.
MATERIALS AND METHODS: Sargassum polycystum was extracted with 95% ethanol and further fractionated with hexane, ethyl acetate and water. The ethanolic crude extract and its fractionated extracts were tested for their potential to act as antimelanogenesis or skin-whitening agents by their abilities to inhibit tyrosinase activity in the cell-free mushroom tyrosinase assay and cellular tyrosinase derived from melanin-forming B16F10 murine melanoma cells. The tyrosinase inhibitory activity was correlated to the inhibition of melanin production in α-MSH-stimulated and unstimulated B16F10 cells.
RESULTS: Sargassum polycystum ethanolic extract and its fractions had little or no inhibitory effect on mushroom tyrosinase activity. However, when tested on cellular tyrosinase, the ethanolic extract and its non-polar fraction, hexane fraction (SPHF), showed significant inhibition of cellular tyrosinase activity. In parallel to its cellular tyrosinase inhibitory activity, SPHF was also able to inhibit basal and α-MSH-stimulated melanin production in B16F10 cells.
CONCLUSIONS: Our findings showed that (i) cellular tyrosinase assay is more reliable than mushroom tyrosinase assay in the initial testing of potential antimelanogenesis agents and, (ii) SPHF inhibited melanogenesis by inhibiting cellular tyrosinase activity. SPHF may be useful for treating hyperpigmentation and as a skin-whitening agent in cosmetics industry.
Fulltext Development and evaluation of polymerase chain reaction assay to detect Burkholderia genus and to differentiate the species in clinical specimens

Suppiah J, Thimma JS, Cheah SH, Vadivelu J

FEMS Microbiol Lett, 2010 May;306(1):9-14.
PMID: 20345378 DOI: 10.1111/j.1574-6968.2010.01923.x

Molecular-based techniques are becoming desirable as tools for identification of infectious diseases. Amongst the Burkholderia spp., there is a need to differentiate Burkholderia pseudomallei from Burkholderia cepacia, as misidentification could lead to false treatment of patients. In this study, conventional PCR assay targeting three genes was developed. Primers were designed for the amplification of Burkholderia genus-specific groEL gene, B. pseudomallei-specific mprA gene and B. cepacia-specific zmpA gene. The specificity and sensitivity of the assay was tested with 15 negative control strains and 71 Burkholderia spp. isolates including positive controls B. pseudomallei K96243 and ATCC B. cepacia strain. All B. pseudomallei strains were positive for groEL (139 bp) and mprA (162 bp), indicating a sensitivity of 100%. All B. cepacia strains produced amplicons for detection of groEL and zmpA (147 bp). Specificity using negative strains was 100%. In this study, a PCR assay specific for the detection of Burkholderia spp. and differentiation of the genus B. pseudomallei and B. cepacia was developed. The conventional assay has to be performed separately for each species due to the similar size of the PCR products amplified. This format may therefore be recommended for use as a diagnostic tool in laboratories where real-time PCR machines are not available. However, the real-time PCR was able to detect and differentiate the genus and species in single duplex assay.
Development of an indirect enzyme immunoassay using monoclonal antibodies for the measurement of 17alpha-hydroxyprogesterone in human serum

Chong H, Cheah SH, Ragavan M, Johgalingam VT

J Immunoassay Immunochem, 2009;30(2):166-79.
PMID: 19330642 DOI: 10.1080/15321810902782863

An indirect enzyme immunoassay for the measurement of total 17alpha-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17alpha-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.
Generation and characterization of monoclonal antibodies against prostate-specific antigen

Karuppaiya A, Cheah SH, Mohd S, Kamal WH, Zulkifli MH

Hybridoma (Larchmt), 2009 Apr;28(2):133-7.
PMID: 19249990 DOI: 10.1089/hyb.2008.0085

Prostate-specific antigen (PSA) is widely used as a diagnostic marker for the detection of prostate cancer in men. We have generated stable hybridomas producing specific monoclonal antibodies (MAbs) of the IgG class against PSA from fusions of splenocytes from immunized mice with myeloma cells. The hybridomas were adapted into serum-free media and cultured in CELLine CL-1000 bioreactors to produce milligram quantities of MAbs. Cross-reactivity study demonstrated that all the MAbs reacted did not cross-react with several other types of tumor antigens. Two of the MAbs were successfully radiolabeled by the iodogen method. The (125)I-labeled MAbs demonstrated strong binding to PSA on the surface of the LNCaP cells (Kd of 1.16 x 10(-9) M and 1.4 x 10(-9) M). Thus the (125)I-labeled MAbs retained their immunoreactivity and possessed high affinity and is potentially useful for binding to tumor cells. In conclusion, the MAbs can be used to develop radioimmunodiagnostic, radioimaging, and immunohistochemistry techniques for the early detection and treatment of prostate cancer.
Production and characterization of monoclonal antibodies against 17alpha-hydroxyprogesterone

Chong H, Cheah SH, Ragavan M, Johgalingam VT

Hybridoma (Larchmt), 2006 Feb;25(1):34-40.
PMID: 16475880

Hybridomas secreting monoclonal antibodies (MAbs) against 17alpha-hydroxyprogesterone (17OHP) have been generated. These MAbs are highly specific and have an affinity of 7-12 x 10(7) M(1). The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma P3X63 Ag8.653 cells. The antigen used for immunization was 17OHP conjugated to bovine serum albumin (17OHP:BSA). Fused cells were plated and cloned in 96-well microtiter plates. Wells containing hybridomas were screened simultaneously for specific gamma globulin (IgG) and anti-17OHP activity using an enzyme-linked immunosorbent assay (ELISA)-based method, which is faster than the conventional radioimmunoassay (RIA) screening procedure. Limiting dilution methods were used to obtain single hybridoma clones producing MAb. The stable hybridomas secreting anti-17OHP MAbs were expanded into bioreactors or ascites fluid for large-scale production of the required antibodies. These MAbs will be used in the formulation of a 17OHP assay kit to screen for congenital adrenal hyperplasia (CAH) in local newborn human population.
Effects of fasting in Ramadan on tropical Asiatic Moslems

Husain R, Duncan MT, Cheah SH, Ch'ng SL

Br J Nutr, 1987 Jul;58(1):41-8.
PMID: 3620437

1. Anthropometric variables, resting heart rate and respiratory gas exchange were measured in twelve male and nine female Asiatic adult Moslems during the month of Ramadan, the week before and the month after Ramadan. 2. Energy intakes were estimated from dietary recall during fasting and non-fasting conditions. 3. Both male and female subjects experienced a decrease in body mass with the reduction in energy intake during fasting. Males experienced a greater reduction than females in resting heart rate; females lost more body-weight and subcutaneous fat than males. 4. Urine output and fluid intake were measured in twelve male subjects for 1 d during each week of fasting and 1 d during the pre-fasting control period. Among the subjects examined, the Ramadan regimen did not result in changes in the pattern of fluid exchange.
Effect of altered eating pattern on serum fructosamine: total protein ratio and plasma glucose level

Ch'ng SL, Cheah SH, Husain R, Duncan MT

Ann Acad Med Singap, 1989 May;18(3):326-7.
PMID: 2774480

The effect of alteration of eating pattern during Ramadan on body mass index (BMI), serum fructosamine: total protein ratio (F/TP), and glucose level in 18 healthy male Asiatic Moslems were studied. The results showed a significant decrease (p less than 0.025) in F/TP at the second week of Ramadan in 11 subjects who experienced continuous decrease in BMI throughout Ramadan. The remaining 7 subjects showed no significant changes in BMI and F/TP. No evidence of hypoglycaemia was observed in the subjects during the study. Serum fructosamine: total protein ratio in subjects with altered eating pattern preferably should be interpreted along with the change in body mass index.
Effects of fasting during Ramadan on urinary excretion in Malaysian Muslims

Cheah SH, Ch'ng SL, Husain R, Duncan MT

Br J Nutr, 1990 Mar;63(2):329-37.
PMID: 2334668

Urine analysis was conducted on male Muslims before, during and after Ramadan. Various changes in urine volume, osmolality, total solute, sodium, potassium, titratable acidity and urea in response to altered feeding and activity regimens were found. There were no detectable levels of ketones, protein, glucose, urobilinogen and haemoglobin. It was concluded that the body adapted to fasting during Ramadan and that there were no adverse effects on renal function.
Fulltext First Report of Eurycoma longifolia Jack Root Extract Causing Relaxation of Aortic Rings in Rats

Tee BH, Hoe SZ, Cheah SH, Lam SK

Biomed Res Int, 2016;2016:1361508.
PMID: 27800486 DOI: 10.1155/2016/1361508

Although Eurycoma longifolia has been studied for erectile function, the blood pressure- (BP-) lowering effect has yet to be verified. Hence, this study aims at investigating the BP-lowering properties of the plant with a view to develop an antihypertensive agent that could also preserve erectile function. Ethanolic root extract was partitioned by hexane, dichloromethane (DCM), ethyl acetate, butanol, and water. The DCM fraction, found to be potent in relaxing phenylephrine- (PE-) precontracted rat aortic rings, was further purified by column chromatography. Subfraction DCM-II, being the most active in relaxing aortae, was studied for effects on the renin-angiotensin and kallikrein-kinin systems in aortic rings. The effect of DCM-II on angiotensin-converting enzyme (ACE) activity was also evaluated in vitro. Results showed that DCM-II reduced (p < 0.05) the contractions evoked by angiotensin I and angiotensin II (Ang II). In PE-precontracted rings treated with DCM-II, the Ang II-induced contraction was attenuated (p < 0.05) while bradykinin- (BK-) induced relaxation enhanced (p < 0.001). In vitro, DCM-II inhibited (p < 0.001) the activity of ACE. These data demonstrate that the vasodilatory effect of DCM-II appears to be mediated via inhibition of Ang II type 1 receptor and ACE as well as enhancement of Ang II type 2 receptor activation and BK activity.
The effects of oestradiol and relaxin on extensibility and collagen organisation of the pregnant rat cervix

Cheah SH, Ng KH, Johgalingam VT, Ragavan M

J Endocrinol, 1995 Aug;146(2):331-7.
PMID: 7561646

The effects of exogenously introduced oestradiol-17 beta (E) and relaxin (RLX) on cervical extensibility and collagen organisation were tested in rats ovariectomised in late pregnancy. When the cervices were stretched in vitro by 1 mm increments, it was found that those from rats given E alone generated significantly higher tensions than those from control rats, while cervices from rats given both E and RLX had tensions similar to controls. Examination of cervical sections under the light microscope and ultra-thin sections under the electron microscope showed that the collagen fibres in the cervices from E-treated rats were highly organised, whereas those from animals given E+RLX and control animals were disorganised and dispersed. It was concluded that E decreased cervical extensibility, while RLX counteracted the effect of E to maintain a soft and easily extensible cervix.
Fulltext Effects of Root Extracts of Eurycoma longifolia Jack on Corpus Cavernosum of Rat

Tee BH, Hoe SZ, Cheah SH, Lam SK

Med Princ Pract, 2017;26(3):258-265.
PMID: 28226311 DOI: 10.1159/000464363

OBJECTIVE: This study was conducted to investigate the mechanisms of action of Eurycoma longifolia in rat corpus cavernosum.
MATERIALS AND METHODS: Tincture of the roots was concentrated to dryness by evaporating the ethanol in vacuo. This ethanolic extract was partitioned into 5 fractions sequentially with hexane, dichloromethane (DCM), ethyl acetate, butanol, and water. The corpus cavernosum relaxant activity of each fraction was investigated. The DCM fraction which showed the highest potency in relaxing phenylephrine-precontracted corpora cavernosa was purified by column chromatography. The effects of the most potent DCM subfraction in relaxing phenylephrine-precontracted corpora cavernosa, DCM-I, on angiotensin I- or angiotensin II-induced contractions in corpora cavernosa were investigated. The effects of DCM-I pretreatment on the responses of phenylephrine-precontracted corpora cavernosa to angiotensin II or bradykinin were also studied. An in vitro assay was conducted to evaluate the effect of DCM-I on angiotensin-converting enzyme activity.
RESULTS: Fraction DCM-I decreased the maximal contractions (100%) evoked by angiotensin I and angiotensin II to 30 ± 14% and 26 ± 16% (p < 0.001), respectively. In phenylephrine-precontracted corpora cavernosa, DCM-I pretreatment caused angiotensin II to induce 82 ± 27% relaxation of maximal contraction (p < 0.01) and enhanced (p < 0.001) bradykinin-induced relaxations from 47 ± 8% to 100 ± 5%. In vitro, DCM-I was able to reduce (p < 0.001) the maximal angiotensin-converting enzyme activity to 78 ± 0.24%.
CONCLUSION: Fraction DCM-I was able to antagonize angiotensin II-induced contraction to cause corpus cavernosum relaxation via inhibition of angiotensin II type 1 receptor and enhance bradykinin-induced relaxation through inhibition of angiotensin-converting enzyme.
Fulltext Ventilatory function in Malay Muslims during normal activity and the Ramadan fast

Duncan MT, Husain R, Raman A, Cheah SH, Ch'ng SL

Singapore Med J, 1990 Dec;31(6):543-7.
PMID: 2281349

Pulmonary function parameters were examined in a Malay Muslim population during normal activity and Ramadan fasting conditions. The validity of employing various lung function prediction formulae for the subjects was also assessed. Present findings indicate that the water deprivation regime and resultant dehydration during Ramadan did not cause significant changes in ventilatory functions. Although pulmonary prediction formulae based on Caucasian and African populations were inapplicable to the subjects examined, the equations derived from the neighbouring populations in Singapore could be employed.
Fulltext Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system

Dharshanan S, Chong H, Cheah SH, Zamrod Z

Cytotechnology, 2014 Aug;66(4):625-33.
PMID: 23881539 DOI: 10.1007/s10616-013-9615-x

From our recent publications, it was found that the deimmunization method (Dharshanan et al. (2012) Sci Res Essays 7:2288-2299) should be applied for the development of humanized anti-C2 monoclonal antibody (H1C2 mAb). However, the overlapping-PCR mutagenesis procedure used to insert the variable regions into cloning vectors was laborious and time-consuming. Additionally, the expression of H1C2 mAb in NS0 cells was low in static culture vessels. Therefore H1C2 mAb was redeveloped by deimmunization method with the following modifications in order to optimize the production of H1C2 mAb. First, instead of the overlapping-PCR mutagenesis procedure, synthetic DNA coding the variable regions were used to express the mAb. Second, two expression vectors, pFUSE and UCOE, were used to express H1C2 mAb in NS0 cells and CHO cells in order to investigate the combination that gave the highest number of high producing stable clones. This will provide the highest chance of finding clones with the requisite high productivity and stability required for manufacturing. We found that transfection of UCOE in CHO cells generated the highest number of high producing stable clones. To our knowledge, this is the first time that H1C2 mAb has been expressed in CHO cells.
Fulltext Culture of low passage colorectal cancer cells and demonstration of variation in selected tumour marker expression

Arul M, Roslani AC, Ng CL, Cheah SH

Cytotechnology, 2014 May;66(3):481-91.
PMID: 23824584 DOI: 10.1007/s10616-013-9600-4

There is increasing evidence that a tumour comprises of heterogeneous population of cells. Thus, studying homogenous cell lines in vitro may yield results that are not reflective of the true situation in a tumour and studying low passage cell lines maintained in a heterogeneous population before they transform away from the original state may provide a more complete picture of colorectal cancer. A method was developed to isolate and establish low passage colorectal cancer cell lines from tumour biopsies. The media contents, combination of antimicrobials and specimen collection and transport conditions employed, successfully eliminated microbial contamination which is frequently present in samples obtained from the gastrointestinal tract. A variety of growth forms indicating a heterogeneous mixture of cells was seen in the initial cultures. Using fluorescence immunocytochemistry, primary tumour cultures were shown to variably express selected tumour markers, carcinoembryonic antigen and C2 antigen. These low passage cell lines growing in a heterogeneous environment would more closely reflect the characteristics of the cells of the original tumour.
Fulltext King cobra (Ophiophagus hannah) venom L-amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumor growth in a tumor xenograft mouse model

Lee ML, Fung SY, Chung I, Pailoor J, Cheah SH, Tan NH

Int J Med Sci, 2014;11(6):593-601.
PMID: 24782648 DOI: 10.7150/ijms.8096

King cobra (Ophiophagus hannah) venom L-amino acid oxidase (OH-LAAO), a heat stable enzyme, has been shown to exhibit very potent anti-proliferative activity against human breast and lung tumorigenic cells but not in their non-tumorigenic counterparts. We further examine its in vitro and in vivo anti-tumor activity in a human prostate adenocarcinoma (PC-3) model. OH-LAAO demonstrated potent cytotoxicity against PC-3 cells with IC50 of 0.05 µg/mL after 72 h incubation in vitro. It induced apoptosis as evidenced with an increase in caspase-3/7 cleavages and an increase in annexin V-stained cells. To examine its in vivo anti-tumor activity, we treated PC-3 tumor xenograft implanted subcutaneously in immunodeficient NU/NU (nude) mice with 1 µg/g OH-LAAO given intraperitoneally (i.p.). After 8 weeks of treatment, OH-LAAO treated PC-3 tumors were markedly inhibited, when compared to the control group (P <0.05). TUNEL staining analysis on the tumor sections showed a significantly increase of apoptotic cells in the LAAO-treated animals. Histological examinations of the vital organs in these two groups showed no significant differences with normal tissues, indicating no obvious tissue damage. The treatment also did not cause any significant changes on the body weight of the mice during the duration of the study. These observations suggest that OH-LAAO cytotoxic effects may be specific to tumor xenografts and less to normal organs. Given its potent anti-tumor activities shown in vitro as well as in vivo, the king cobra venom LAAO can potentially be developed to treat prostate cancer and other solid tumors.

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