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S Y Chan

Publications and source records attributed to S Y Chan.

At least 73 records · Page 4Linked to original sources
PubMed2000

An efficient method of generating transgenic mice by pronuclear microinjection.

Genetic transformation of mice using pronuclear microinjection was demonstrated by a number of groups in rapid succession in the early 1980's. Since that time, studies using transgenic animals have produced major advances in biomedical sciences and molecular genetics. More important, it is possible to study the molecular basis for tissue and stage-specific expression of genes. We recently used this method to generate transgenic mice. DNA fragment (transgene) was injected into the pronucleus of one-cell embryos. We describe this simplified protocol, which is reliable. With the use of buffered medium M2 for the whole process, it is not mandatory to have a CO2 incubator.

Animals↗
PubMed2000

Investigating plant-based medicines for wound healing with the use of cell culture technologies and in vitro models: a review.

INTRODUCTION: Cell culture and molecular technologies are basic yet sophisticated research tool used to investigate plant-based medicine for wound healing. METHODS: Cell viability and proliferation assay is used to determine whether there are any positive effects and to discover what is the limiting cytotoxic concentration in vitro. The scratch technique, fibroblast-populated collagen lattices and aortic rings embedded gels are used as the in-vitro models of wound re-epithelialization, contraction and angiogenesis. The immunofluorescence, immunoblotting and organotypic culture can be used to detect expression of specific proteins that are modulated by plant extracts during the wound healing process. MAIN FINDINGS: Given the dynamics of the wound healing process, cell culture and molecular technologies are advantageous in providing us with detailed studies and analysis of each intricate process. CONCLUSION: The scientific approaches for the study of traditional plant-based remedies for wound healing will provide us an important platform for rigorous testing and evaluation of their clinical efficacy based on accepted rules of evidence.

Cell Survival↗
PubMed2000

Differential morphometric and ultrastructural remodelling in the left atrium and left ventricle in rapid ventricular pacing-induced heart failure.

BACKGROUND: Heart failure induced by rapid ventricular pacing (RVP) is associated with left atrial (LA) but not left ventricular (LV) hypertrophy. OBJECTIVE: To determine whether differences in wall tension correlate with the differential ultrastructural remodelling in the LA and LV chambers, changes in ultrastructure, systolic function and wall tension (an index of wall stress) were compared in dogs after RVP (n=7) and with no RVP (n=9). RESULTS: Compared with dogs with no RVP (controls), dogs with RVP had increased collagen volume fraction (5.3% versus 8.3%), myocyte cross-sectional area (245 versus 366 microm(2)) and hydroxyproline (222 versus 323 microg/mg protein) in the LA (all P<0.05), but not in the LV. The increase in systolic wall tension produced by RVP was greater in the LA (five versus 43 units, P<0.0004) than in the LV (227 versus 290 units, P<0.01) chambers and correlated closely with the collagen volume fraction (r=0.87), which in turn correlated with myocyte cross-sectional area (r=0.98). In the left atrium, wall tension correlated with wall stress (r=0.99). CONCLUSIONS: The results suggest that differential wall tension may provide the stimulus for differential ultrastructural remodelling (with more hypertrophy and collagen) between LA and LV chambers in RVP-induced cardiomyopathy.

Animals↗
PubMed1999

Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for Th1 in the disease process.

BACKGROUND: Transient depletion of CD4+ T cells in cardiac allograft recipients prolongs allograft survival; however, grafts exhibit signs of chronic rejection characterized by collagen deposition and neointima development. Although it is believed that Th1 cells promote acute graft rejection, the role of these cells in chronic rejection remains unclear. Hence, our study evaluated whether Th1 cells are associated with the development of chronic cardiac allograft rejection. METHODS: Splenocytes obtained from C57BL/6 recipients bearing BALB/c hearts with signs of chronic rejection were adoptively transferred into C57BL/6 SCID cardiac allograft recipients. As a measure of Th1 function, interferon-y production was determined after restimulation of recipient splenocytes with donor alloantigens. RESULTS: Transfer of splenocytes in SCID allograft recipients resulted in accelerated chronic rejection in the majority of mice. Characterization of these cells before transfer revealed hyporesponsive Th1 function. However, donor-specific proliferative responses and precursor interleukin-2 producing helper and cytotoxic T lymphocyte frequencies were comparable to that of naive splenocytes. Further, splenocytes obtained from SCID recipients with advanced signs of chronic rejection remained deficient in Th1 function, suggesting that Th1 are not involved in this disease process. This possibility was further supported by the development of chronic rejection in IL-12 knockout recipients. Finally, when splenocytes used for adoptive transfer retained Th1 function, transfer of these cells into SCID recipients resulted in acute allograft rejection. CONCLUSIONS: We have established a model in which the mediators of chronic rejection may be further explored. In this system, the absence rather than the presence of donor-reactive Th1 is associated with chronic rejection. These data indicate that Th1-independent effector mechanisms are responsible for chronic rejection in this model.

Adjuvants, Immunologic↗
PubMed1999

Interleukin-12 (IL-12)-driven alloimmune responses in vitro and in vivo: requirement for beta1 subunit of the IL-12 receptor.

BACKGROUND: Interleukin-12 (IL-12) mediates its biologic activities via binding high-affinity receptors on T and natural killer cells. Although emphasis has been placed on the requirement for IL-12Rbeta2 in IL-12 bioactivity, the role of IL-12Rbeta1 is less well defined. The current study evaluated the effects of exogenous IL-12 on alloantigen-specific immune responses and determined the requirement for IL-12Rbeta1 in IL-12-mediated alloimmunity. METHODS: The mouse heterotopic cardiac transplant model was employed to evaluate the effects of IL-12 on alloantigen-specific immune responses in vivo. In addition, IFN-gamma production in mixed lymphocyte cultures (MLC) supplemented with IL-12 was measured to assess the effects of IL-12 on Th1 function in vitro. Mice deficient in IL-12Rbeta1 (IL-12Rbeta1-/-) were used to determine the requirement for this receptor component in IL-12-driven alloimmune responses. RESULTS: Addition of IL-12 to MLC consisting of wild-type splenocytes enhanced alloantigen-specific proliferative responses and Th1 development. In contrast, IL-12 did not alter these in vitro immune parameters in IL-12Rbeta1-/- MLC. Treatment of wild-type cardiac allograft recipients with IL-12 resulted in high concentrations of serum interferon-gamma (IFN-gamma) and a 10-fold increase in IFN-gamma production by recipient splenocytes after restimulation in vitro. However, this fulminate Th1 response did not accelerate allograft rejection. Importantly, IL-12 had no effect on serum IFN-gamma or in vivo priming of Thl in IL-12Rbeta1-/- recipients. Finally, administration of IL-12 to WT allograft recipients resulted in a bimodal alloantibody response: antibody production was suppressed at high doses of IL-12, and enhanced at lower doses. CONCLUSIONS: IL-12 markedly enhances alloantigen-specific immune function; however, these exaggerated Th1-driven responses do not culminate in accelerated allograft rejection. Further, these data indicate that IL-12Rbeta1 is essential for the enhancement of both in vitro and in vivo alloimmune responses by exogenous IL-12.

Animals↗
PubMed1999

A trans-receptor mechanism for infection of CD4-negative cells by human immunodeficiency virus type 1.

Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4(-) cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2] [3] [4] [5] [6], epithelial cells [5] [7], CD4(-) lymphocytes and thymocytes [8] [9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4(-) cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4(+) cells. This process requires contact between the coreceptor-bearing and CD4(+) cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4(-) cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.

CD4 Antigens↗
PubMed1999

Tissue-specific consequences of the anti-adenoviral immune response: implications for cardiac transplants.

The immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. This would limit the use of adenovirus-mediated gene transfer in disease states in which long-term gene expression is required. While studying the effect of the anti-adenoviral immune response in transplantation, we found that transgene expression persisted in cardiac isografts transfected with an adenovirus encoding beta-galactosidase. Transfected grafts remained free of inflammation, despite the presence of an immune response to the vector. Thus, adenovirus-mediated gene transfer may have therapeutic value in cardiac transplantation and heart diseases. Furthermore, immunological limitations of adenoviral vectors for gene therapy are not universal for all tissue types.

Adenoviridae↗
PubMed1999

Effects of sucrose, citric buffer and glucose oxidase on the stability of captopril in liquid formulations.

OBJECTIVE: To study the effects of sucrose, citric buffer and glucose oxidase on the stability of captopril in liquid formulations. METHOD: Captopril liquid formulations 1 mg/ml were prepared in various concentrations of syrup with and without citric buffer. The liquid formulations were stored in amber glass bottles at 5 degrees C. Samples were removed from these formulations at 0, 1, 2, 3, 4, 7, 10, 15, 20 and 30 days for assay of captopril by a stability-indicating high performance liquid chromatographic method. RESULTS: Our findings indicate that low concentration of citric buffer (0.03 M) had a small stabilising effect on captopril in liquid formulations containing no or low concentration of sucrose (10% w/v). However, in formulations with higher concentrations of sucrose (30 and 85% w/v, respectively), the stabilising effect was not apparent. Captopril in 1 mg/ml formulations containing 0.03 M citric buffer and 10% w/v glucose degraded rapidly in the presence of glucose oxidase. The degradation was particularly evident at higher temperature. Glucose oxidase is an antioxidant, which can extensively reduce oxygen concentration in liquid formulations. CONCLUSION: The stability of captopril is very sensitive to the presence of excipients. Many excipients can function as catalysts in its degradation. Even in the deficiency of oxygen, its degradation can also be substantial with suitable catalysts present in the liquid formulation.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Is mammography of value in women with disseminated cancer of unknown origin?

Mammography is often requested to try to identify occult primary breast carcinoma in women with metastatic cancer of unknown primary site. This study aimed to investigate whether mammography is of use in these patients in identifying the breast as the origin of the metastatic disease. Thirty-one women with a working diagnosis of metastatic cancer underwent mammography in an attempt to determine the primary site. None of these women had a palpable breast mass. The site of presentation, pathological type of tumour, site of origin, and benefit of mammography and mammography-provoked biopsy were clarified for each patient. The patients were also followed up to determine survival. The commonest sites of presentation were lung (45%), lymph nodes (19%) and abdomen (16%). The primary sites of these cancers were identified with confidence in 27 patients (87%). The commonest known primary tumour sites were lung (45%), breast (16%) and ovary (16%). Abnormal mammograms were detected in four patients (13%), but three of these did not have breast cancer. In one, the site of origin remained indeterminate, as either breast or lung. Five (16%) had a confident diagnosis of breast carcinoma; all of these women had normal mammograms. We conclude that mammography in women presenting with metastatic disease from an unknown primary site is unhelpful and is not recommended. Furthermore, we could not demonstrate its value in women presenting with axillary lymphadenopathy.

Adult↗
PubMed1999

Isolation of Helicobacter pylori from vomitus in children and its implication in gastro-oral transmission.

OBJECTIVE: The route of transmission of Helicobacter pylori (H. pylori) is unclear. Gastro-oral transmission via contaminated vomitus has been proposed as an important mode of transmitting H. pylori, especially in children. This pilot study attempted to isolate H. pylori from the vomitus of children. METHODS: Children presenting for evaluation with gastroenteritis-associated vomiting were studied. Fresh vomitus samples were collected for detection of H. pylori by bacteriological culture and polymerase chain reaction, (PCR). A rapid, whole blood test was used to determine the H. pylori status of patients. RESULTS: A total of 18 children with mean age of 6 yr were studied; four had a positive serology test. Among these four children, H. pylori was isolated from vomitus by culture in one child and by PCR in two. An 18-month-old girl with negative serology had H. pylori detected in vomitus by PCR. Six months later, she had seroconversion confirmed, suggesting that she had an acute H. pylori infection on initial presentation. CONCLUSIONS: This is the first study reporting successful isolation of H. pylori from naturally produced vomitus. The result implies that transmission of H. pylori infection by vomitus, especially in children, is possible.

Adolescent↗
PubMed1999

V3 recombinants indicate a central role for CCR5 as a coreceptor in tissue infection by human immunodeficiency virus type 1.

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.

AIDS Dementia Complex↗
PubMed1999

Expression of full length or truncated epidermal growth factor precursor transforms NIH3T3 fibroblasts.

Epidermal growth factor (EGF) is derived from a large precursor (EGFP) of unusual structure. As EGFP remains unprocessed in certain tissues, it is of biological relevance to study its activity. Activation of the EGF receptor by EGF is involved in transformation of NIH3T3 fibroblasts. We isolated clones of NIH3T3 expressing full length, cytoplasmic region deleted or EGF-repeats deleted EGFP. All clones formed colonies in soft agarose and tumors in nude mice. Two clones expressing EGF-repeats deleted EGFP formed more and larger colonies. To conclude, EGFP is biologically active. Deletion of the 8 EGF repeats may enhance anchorage independent growth in NIH3T3.

3T3 Cells↗
PubMed1998

Genetic vaccination-induced immune responses to the human immunodeficiency virus protein Rev: emergence of the interleukin 2-producing helper T lymphocyte.

Rev M10 is a trans-dominant negative inhibitor of HIV replication. Hence, stable transduction of CD4+ T cells with Rev M10 represents a novel gene therapy aimed at inhibiting HIV replication within these cells, thereby slowing the progression of AIDS. However, the immune system may recognize Rev M10 as foreign and target transduced cells for elimination. In the current study, mice were genetically immunized with a plasmid encoding Rev M10, to (1) identify immune parameters that may be induced by Rev M10 gene transfer, (2) determine the impact of repeated introduction of the Rev M10-encoding plasmid on the immune response to the transgene product, and (3) determine if cotransfection with a plasmid encoding TGFbeta1 would suppress the response. Kinetic studies revealed that Rev-specific IL-2-producing helper T lymphocytes (HTLs) appeared following the second genetic immunization, peaked after the third, and persisted at peak levels for at least 6 weeks. Rev-specific HTLs were CD4+, and the development of these cells was ablated by cotransfection with TGFbeta1. Other cytokines were not readily detectable when immune splenocytes were restimulated with Rev in vitro, and Rev-specific IgG antibodies were not present in the sera of these mice. To our knowledge, this represents the first report that genetic immunization with Rev M10 induces an immune response that is dominated by IL-2-producing HTLs. Further, this study demonstrates the potential utility of introducing immunosuppressive genes as a means to control the immune response to foreign transgene products.

Animals↗
PubMed1998

Sex difference in immunostaining of RET in the adult mouse kidney.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which is important for the development of the kidney and the enteric nervous system. During nephrogenesis, c-ret is expressed in the ureteric bud epithelium and later in its derivative, the collecting duct. This takes place during 11-17.5 days post-coitum (d.p.c.) in the mouse and our immunohistochemical study showed that the RET protein co-localized with the transcript. At 18.5 d.p.c. the kidney is fully differentiated. At 18.5 d.p.c., 1 week and 10 weeks old, RET was found in the proximal convoluted tubules, which is formed from the condensed mesenchyme. This suggests that c-ret may also play a role in kidney function. For the 10 weeks old kidney, RET immunostaining in male was concentrated on the basolateral side while female had a stronger staining in the whole cell. Furthermore, cytoplasmic staining was observed in male whereas both cytoplasmic and nuclear staining was found in female. c-ret transcript was detected by RT-PCR, and in situ hybridization showed its expression throughout the kidney. The reason for the sex-specific staining and the role of RET in kidney function remain to be determined.

Animals↗
PubMed1998

Alloantigen-reactive Th1 development in IL-12-deficient mice.

IL-12p70, a 70- to 75-kDa heterodimer consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits, enhances Th1 development primarily by its ability to induce IFN-gamma production by NK and Th1 cells. Although homodimers of the p40 subunit of IL-12 are potent IL-12 receptor antagonists in some systems, we have reported that p40 homodimer may accentuate alloreactive CD8+ Th1 function. To test the role of endogenously produced p40 in alloimmunity, Th1 development was assessed in either IL-12 p35 knockout (p35-/-) mice, the cells of which are capable of secreting p40, or p40 knockout (p40-/-) mice. Compared with IL-12 wild-type controls, splenocytes obtained from both p35-/- and p40-/- mice produced markedly less IFN-gamma after in vitro stimulation with Con A or alloantigens. Interestingly, in vivo-sensitized Th1 were detected in both p35-/- and p40-/- cardiac allograft recipients. However, in vivo Th1 development was enhanced in p35-/- recipients compared with p40-/- animals, suggesting that endogenous p40 produced in p35-/- mice may stimulate alloreactive Th1. Indeed, neutralizing endogenous p40 with anti-IL-12 p40 mAb reduced Th1 development in p35-/- allograft recipients to that seen in p40-/- mice. To determine whether Th1 development that occurred in the absence of IL-12p70 and p40 required IFN-gamma, p40-/- allograft recipients were treated with anti-IFN-gamma mAb. Neutralizing IFN-gamma did not inhibit in vivo Th1 development in p40-/- recipients and resulted in a unique pathology of rejection characterized by vascular thromboses. Collectively, these data suggest that 1) endogenous p40 may substitute for IL-12p70 in alloantigen-specific Th1 sensitization in vivo and 2) in vivo alloreactive Th1 development may occur independent of IL-12 and IFN-gamma, suggesting an alternate Th1-sensitizing pathway.

Animals↗
PubMed1998

Controlled multicentre study of the influence of subcutaneous recombinant human erythropoietin on anaemia and transfusion dependency in patients with ovarian carcinoma treated with platinum-based chemotherapy.

This randomised controlled multicentre trial evaluated the effectiveness of recombinant human erythropoietin (rhEPO) in preventing anaemia and reducing the need for blood or erythrocyte transfusion in 122 ovarian cancer patients receiving platinum-based chemotherapy. The patients were randomly allocated to receive rhEPO 150 U/kg or 300 U/kg subcutaneously, three times a week, or open control. Patients also received up to 6 cycles of carboplatin or cisplatin, alone or in combination with other cytotoxic agents. Intention-to-treat analysis showed that 39.4% of patients in the control group received at least one blood transfusion, compared with 9.2% of patients treated with rhEPO. Patients treated with rhEPO experienced a significantly longer time to first erythrocyte transfusion than the control group and were less likely to experience nadir haemoglobin levels < 10 g/dl (P < 0.001 and < 0.05, respectively). A haemoglobin decrease < 1 g/dl during the first chemotherapy cycle, as well as a low baseline serum erythropoietin concentration, predicted a low transfusion need in rhEPO-treated patients but not in controls. During the study, 103 patients suffered at least one adverse event, but no serious, and only nine non-serious adverse events were considered possibly related to rhEPO therapy. These results indicate that treatment with rhEPO prevents anaemia, it reduces the need for blood or rhEPO erythrocyte transfusion in patients with ovarian cancer receiving platinum-based chemotherapy, and it is well tolerated. A starting dose of 150 U/kg of rhEPO, three times a week, may be recommended.

Anemia↗
PubMed1998

Expression of transforming growth factor alpha and epidermal growth factor receptor in adult polycystic kidney disease.

Adult polycystic kidney disease (APKD) is a common genetic disease with a frequency of 1:1000. Evidence suggests that transforming growth factor alpha (TGF alpha) signaling may contribute to the hyperproliferation of the cystic epithelia in APKD. TGF alpha and epidermal growth factor (EGF) are well known mitogens expressed in the kidney and both exert their biological activities through binding to the same EGF receptor. A transgenic mouse that over-expressed TGF alpha developed renal cysts; raised levels of TGF alpha and EGF receptor mRNA were found in kidneys from two autosomal dominant APKD patients. To study the role of TGF alpha in cyst formation, we analyzed nine anatomically diagnosed adult polycystic kidneys and four normal kidneys using immunohistochemistry. We also traced the possible origins of the cysts by staining with the proximal convoluted tubule (PCT) marker, gp330, and the distal convoluted tubule (DCT) and collecting tubule (CT) marker, peanut agglutinin (PNA). In normal kidneys, TGF alpha protein was concentrated in the DCT and CT and EGF receptor protein in all three tubule types. In the early cysts of APKD, the cystic epithelia showed strong positive staining with TGF alpha, EGF receptor and gp330 but negative with PNA. Strong TGF alpha and EGF receptor staining was also found in the mixture of advanced cysts in the end-stage cystic kidneys although the cysts are likely to be derived from different segment of the renal tubules. This increased TGF alpha and EGF receptor expression in all cases and all types of cysts suggests that autocrine/paracrine stimulation by TGF alpha may be a common mechanism in cyst development in APKD.

Adult↗