MyMedR

Detection of Epstein-Barr virus DNA in nasopharyngeal carcinoma using a non-radioactive digoxigenin-labelled probe

Permeen AM, Sam CK, Pathmanathan R, Prasad U, Wolf H

J Virol Methods, 1990 Mar;27(3):261-7.

The presence of Epstein Barr virus (EBV) DNA in biopsies from the post-nasal space (PNS) of patients suspected of nasopharyngeal carcinoma (NPC) was detected by in situ cytohybridization with an EBV DNA probe labelled with the novel labelling compound digoxigenin. The digoxigenin probe was hybridised to cryostat sections of NPC biopsies and subsequently detected by an enzyme immunoassay procedure. It was found that in situ cytohybridization using the digoxigenin probe was much more rapid and sensitive (96 h compared to five weeks) than the current method of using 3H-labelled probe. Using the digoxigenin EBV probe, it was found that in all the eighteen NPC biopsies tested, EBV DNA was detected in malignant epithelial cells and infiltrating lymphocytes. EBV DNA was also detected in some normal epithelial cells in these NPC biopsies. EBV DNA was not detected in epithelial cells of non-malignant biopsies.

Fulltext Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia

Chan KG, Atkinson S, Mathee K, Sam CK, Chhabra SR, Cámara M, et al.

BMC Microbiol, 2011 Mar 08;11:51.

PMID: 21385437 DOI: 10.1186/1471-2180-11-51

BACKGROUND: Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest.

RESULTS: By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the genera Acinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens.

CONCLUSIONS: Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.

Fulltext Mutational analysis of p53 and RB2/p130 genes in Malaysian nasopharyngeal carcinoma samples: a preliminary report

Hoe SL, Sam CK

Malays J Pathol, 2006 Jun;28(1):35-9.

PMID: 17694957 MyJurnal

This study reports the results of mutation detection of tumour suppressor genes, p53 and RB2/p130 genes in Malaysian nasopharyngeal carcinoma (NPC) studied by PCR-CSGE analysis and direct DNA sequencing method. Frequent sites of mutation in both genes (exons 5-8 of p53 and exons 19-21 of RB2/p130) were examined. Thirty-six NPC blood samples and three NPC cell lines were investigated for the presence of mutations. No mutation of p53 and RB2/p130 genes was identified in any of the blood samples. Nonetheless, there was an identical G-->4 C nucleotide change at codon 280 of p53 gene in all the cell lines. A larger study that includes biopsy tissues should be carried out to provide a more in-depth look into the pathogenesis of NPC in Malaysia.

Quantification of Epstein-Barr virus DNA load, interleukin-6, interleukin-10, transforming growth factor-beta1 and stem cell factor in plasma of patients with nasopharyngeal carcinoma

Tan EL, Selvaratnam G, Kananathan R, Sam CK

BMC Cancer, 2006;6:227.

PMID: 16995954

Nasopharyngeal carcinoma (NPC) is a common epithelial neoplasm among the Chinese populations in Southern China and South East Asia. Epstein-Barr virus (EBV) is known to be an important etiologic agent of NPC and the viral gene products are frequently detected in NPC tissues along with elevated antibody titres to the viral proteins (VCA and EA) in a majority of patients. Elevated plasma EBV DNA load is regarded as an important marker for the presence of the disease and for the monitoring of disease progression. However, other serum/plasma parameters such as the levels of certain interleukins and growth factors have also been implicated in NPC. The objectives of the present study are, 1) to investigate the correlations between plasma EBV DNA load and the levels of interleukin (IL)-6, IL-10, TGF-beta1 and SCF (steel factor) and 2) to relate these parameters to the stages of NPC and the effect of treatment.

Epstein-Barr virus serology in the diagnosis of nasopharyngeal carcinoma

Wong MM, Lye MS, Cheng HM, Sam CK

Asian Pac J Allergy Immunol, 2005 Mar;23(1):65-7.

PMID: 15997877

The antibody levels to viral capsid antigen (VCA) and early antigen (EA) of Epstein-Barr virus (EBV) in 164 nasopharyngeal carcinoma (NPC) patients from Sarawak, East Malaysia were significantly higher than those in 147 sex, age and ethnically matched healthy controls. As diagnostic markers of NPC, IgG/VCA at reciprocal titers > or =160 was the most sensitive (89%, with 98% specificity), while IgA/EA at > or =5 was the most specific (100%) but the least sensitive (75%). The sensitivity and specificity of IgA/VCA at reciprocal titers > or =10 were 84% and 97%. IgA/VCA has an advantage over IgG/VCA despite the slightly lower sensitivity due to its consistently more distinct fluorescence reaction. The sensitivity and specificity can be marginally improved by a combination of two tests.

Analyses of Epstein-Barr virus latent membrane protein-1 in Malaysian nasopharyngeal carcinoma: high prevalence of 30-bp deletion, Xho1 polymorphism and evidence of dual infections

Tan EL, Peh SC, Sam CK

J Med Virol, 2003 Feb;69(2):251-7.

PMID: 12683415

Nasopharyngeal carcinoma, a malignancy associated closely with Epstein-Barr virus (EBV), is prevalent among Chinese of Southern China origin. Epidemiological studies indicate a high prevalence of EBV in Asia with viral isolates having typical characteristics of the putative viral oncogene, latent membrane protein 1 (LMP-1), such as the loss of the Xho1 restriction site in Exon 1 and the 30-bp deletion in Exon 3. The EBV LMP-1 gene from throat washings of 120 nasopharyngeal carcinoma patients and 14 healthy individuals were analyzed. Similar analyses were also carried out on 30 and 12 postnasal space biopsies from nasopharyngeal carcinoma patients and healthy individuals, respectively. The 30-bp deletion was detected in 20% of nasopharyngeal carcinoma throat washes and in 100% of nasopharyngeal carcinoma postnasal space biopsies. Interestingly, 16% of the nasopharyngeal carcinoma biopsies possessed both the deleted and the undeleted variants, suggestive of dual infections. The notion of dual infections in nasopharyngeal carcinoma was further supported by the coexistence of both "F" and "f" (BamH1F region) EBV variants in 11% of the nasopharyngeal carcinoma biopsies. All of the throat washes and biopsies from the healthy controls showed the undeleted variant. The loss of the Xho1 restriction site was found with higher frequency both in throat washes and biopsies from patients with nasopharyngeal carcinoma. The discrepancy in the frequency of the 30-bp deletion between throat washes (20%) and postnasal space biopsies (100%) was an indication that this deletion is specific for viral isolates from primary tumour sites.

Fulltext The relevance of specific serum IgG, IgG4 and IgE in the determination of shrimp and crab allergies in Malaysian allergic rhinitis patients

Yeoh SM, Sam CK

Asian Pac J Allergy Immunol, 2001 Mar;19(1):7-10.

PMID: 11495303

The significance of food specific serum IgG4 antibody in food allergy is unclear and this led us to investigate the relevance of specific IgG4, along with IgG and IgE antibodies to two common food allergens in Malaysia. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum antibodies in 143 allergic rhinitis patients' sera, of which 47 were from patients with clinical indication of shrimp allergy, 46 with clinical indication of crab allergy and 50 without indication to either allergy. Clinical indication of allergy was based on answers to a questionnaire or results of the skin prick test. We found that the elevation of specific IgE or IgG4 is associated with shrimp and crab allergies but elevation of specific IgG is not associated with either allergy. However, the clinical utility of elevated specific IgG and IgG4 levels is pending further investigation.
Study site: Allergic rhinitis clinic, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia

Heat inactivation of serum potentiates anti-cardiolipin antibody binding in ELISA

Cheng HM, Ngeow YF, Sam CK

J Immunol Methods, 1989 Nov 30;124(2):235-8.

PMID: 2600427

Heat treatment of sera at 56 degrees C for 30 min results in positive ELISA reactions for anti-cardiolipin antibody (aCL) in sera that had undetectable or low levels of aCL before heat inactivation. The positive, potentiated reactivity of the heated sera in the aCL ELISA could be inhibited with the cardiolipin antigen and was abolished by prior IgG depletion using staphylococcal protein A. The heat-potentiating effect of aCL binding in ELISA was evident in both normal human sera and clinical sera including sera from patients with systemic lupus erythematosus and syphilis.

D1S80 (pMCT118) allele frequencies in a Malay population sample from Malaysia

Koh CL, Lim ME, Ng HS, Sam CK

Int J Legal Med, 1997;110(1):39-40.

PMID: 9081241

The D1S80 allele frequencies in 124 unrelated Malays from the Malaysian population were determined and 51 genotypes and 19 alleles were encountered. The D1S80 frequency distribution met Hardy-Weinberg expectations. The observed heterozygosity was 0.80 and the power of discrimination was 0.96.

Biological properties of TW01 cells expressing latent membrane protein-1 gene of EBV-derived from nasopharyngeal carcinoma cells at different stages of malignancy

Tan EL, Sam CK

Exp Oncol, 2007 Sep;29(3):166-74.

PMID: 18004239

Epstein-Barr virus (EBV), a human gammaherpesvirus is intimately associated with nasopharyngeal carcinoma (NPC), with the incidence of the virus detected in malignant tissues being close to 100% in NPC endemic areas. The viral latent gene, latent membrane protein 1 (LMP1), has all the typical characteristics of an oncogene and extensive studies have shown beyond doubt its abilities in cellular transformation giving rise to malignant phenotypes. The present study compares the gene sequence and biological properties of LMP1 gene derived from two patients with different stages of NPC--one presented with dysplastic, pre-malignant lesion and the other with malignant lesion.

Fulltext Human leukocyte class I antigen alleles A2 and A11 are not associated with nasopharyngeal carcinoma in West Malaysia

Lee LK, Tan EL, Gopala K, Sam CK

Singapore Med J, 2007 Jul;48(7):632-4.

PMID: 17609824

Nasopharyngeal carcinoma (NPC) is the second most common cancer among Malaysian Chinese males. We determined the frequencies of 17 human leukocyte antigens (HLA), HLA-A and HLA-B, alleles in 88 Malaysian Chinese with NPC.

Fulltext Evaluation of plasma Epstein-Barr virus DNA load as a prognostic marker for nasopharyngeal carcinoma

Tan EL, Looi LM, Sam CK

Singapore Med J, 2006 Sep;47(9):803-7.

PMID: 16924363

Nasopharyngeal carcinoma (NPC) is an important cancer in Malaysia and is one of the major causes of cancer mortality in this country. This study evaluates the diagnostic and prognostic values in the quantitative relationship between the cell-free Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) load and the tumour burden.

Bacterial immunity and immunogenesis of normal human salivary IgA and serum IgG2 antiphospholipid autoantibody: a link?

Cheng HM, Sam CK

Immunol Lett, 1990 Oct;26(1):7-10.

PMID: 2276764

The anti-phospholipid antibody (aPL) in 26 heat-inactivated normal human sera (NHS) was tested for IgG subclass in ELISA. The specific antibody in NHS included all four IgG antibody subclasses, as well as IgA. The incidence of IgG subclasses ranged from 50% (13/26) for IgG1 to 92% (24/26) for IgG2. Specific IgA anti-phospholipid antibody (aPL) was detected by ELISA in 38% (28/73) of normal human saliva. The salivary IgA aPL bound preferentially to anionic phospholipids including cardiolipin, phosphatidylserine and phosphatidic acid but not to phosphatidylcholine or sphingomyelin. Unlike aPL in normal human sera, aPL in saliva was predominantly not associated with the previously described heat-labile inhibitor of aPL. This may indicate a role of salivary IgA aPL in local immunity by binding to cross-reactive bacterial cell surface components including phospholipids.

IgA/VCA as a follow-up marker in the monitoring of nasopharyngeal carcinoma

Sam CK, Abu-Samah AJ, Prasad U

Eur J Surg Oncol, 1994 Oct;20(5):561-4.

PMID: 7926060

Titers of IgA/VCA from 92 nasopharyngeal carcinoma (NPC) patients were monitored for 3 to 11 years from the time of diagnosis. The fluctuations in the IgA/VCA titers during follow-up did not correlate with the clinical status of the patients, suggesting that IgA/VCA is of marginal significance in the monitoring of NPC patients during follow-up. In addition, the frequency of recurrence of NPC was independent of presence or absence of elevated IgA/VCA at diagnosis.

Serum IgA cross-reactivity between glycine-alanine repeat sequence of EBNA-1 and keratin or collagen in nasopharyngeal carcinoma

Mathew A, Cheng HM, Sam CK, Prasad U

Clin. Immunol. Immunopathol., 1994 May;71(2):164-8.

PMID: 7514112

Inhibition studies were carried out to study possible cross-reactivity between a peptide fragment of the Epstein-Barr virus nuclear antigen, EBNA-1, and keratin/collagen. The 20-amino acid peptide (pAG), derived from a glycine-alanine repeat region of EBNA-1, uniquely makes up about one-third of the viral protein and is a dominant IgA antigenic epitope in patients with nasopharyngeal carcinoma (NPC). A small percentage of normal human sera (NHS) also binds pAG and this reactivity is examined in this study. Ten percent (2/20) and 13.4% (2/15) of IgA-pAG-positive NPC sera and NHS, respectively, were significantly inhibited by keratin in a competitive ELISA system. Conversely, 31.6% (6/19) and 30.8% (4/13) of IgA-keratin-positive NPC sera and NHS, respectively, were significantly inhibited by pAG. This indicated minimum cross-reactivity between IgA serum antibodies to EBNA-1 and keratin. Using collagen as inhibitor, none of 18 and only 2/13 IgA-pAG-positive NPC sera and NHS, respectively, were inhibited. In the collagen ELISA system, only 2/19 (10.5%) and 4/25 (16%) of IgA-collagen-positive NPC sera and NHS, respectively, were inhibited with pAG. Therefore, cross-reactivity with collagen was also low. IgA-pAG-positive NHS may therefore not be a false positive phenomenon, but whether it may represent an early serological profile related to NPC carcinogenesis remains to be determined.

Serum and salivary IgA antibodies against a defined epitope of the Epstein-Barr virus nuclear antigen (EBNA) are elevated in nasopharyngeal carcinoma

Foong YT, Cheng HM, Sam CK, Dillner J, Hinderer W, Prasad U

Int J Cancer, 1990 Jun 15;45(6):1061-4.

PMID: 1693600

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.

Improved sensitivity of detection by avidin-biotin complex (ABC) immunocytochemistry in Epstein-Barr virus serology

Thoe SY, Sam CK, Cheng HM, Prasad U

J Med Virol, 1989 Dec;29(4):311-4.

PMID: 2559955

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.

Elevated secretory IgA antibodies to Epstein-Barr virus (EBV) and presence of EBV DNA and EBV receptors in patients with cervical carcinoma

Se Thoe SY, Wong KK, Pathmanathan R, Sam CK, Cheng HM, Prasad U

Gynecol Oncol, 1993 Aug;50(2):168-72.

PMID: 8397152

Epstein-Barr virus (EBV) receptors (EBV/C3d receptors) were detected, using the monoclonal antibody HB5, on 23 ectocervical and 5 endocervical biopsies of the uterine cervix. Elevated IgA titers against the viral capsid antigen and early antigen of EBV were also found in the cervical secretions from cervical carcinoma patients (83%), compared with samples from patients with cervical intraepithelial neoplasia (75%), herpes simplex virus-infected patients (0%), and gynecologic patients with nonmalignant conditions (0%). EBV DNA was present in 63% of cervical carcinoma biopsies detected by in situ hybridization. These observations suggest a positive association between EBV and carcinoma of the cervix.

Screening for nasopharyngeal carcinoma with an ELISA using the Epstein-Barr virus nuclear antigen, EBNA 1: a complementary test to the IgA/VCA immunofluorescence assay

Cheng HM, Foong YT, Mathew A, Sam CK, Dillner J, Prasad U

J Virol Methods, 1993 Apr;42(1):45-51.

PMID: 7686558

An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.

Linear epitopes of the replication-activator protein of Epstein-Barr virus recognised by specific serum IgG in nasopharyngeal carcinoma

Cheng HM, Foong YT, AbuSamah AJ, Dillner J, Sam CK, Prasad U

Cancer Immunol Immunother, 1995 Apr;40(4):251-6.

PMID: 7750123

The linear antigenic epitopes of the Epstein-Barr virus replication activator protein (ZEBRA), recognised by specific serum IgG in nasopharyngeal carcinoma (NPC), were determined. This was achieved by synthesizing the entire amino acid sequence of ZEBRA as a set of 29, 22-residue peptides with an overlap of 14 amino acids. The ZEBRA peptides were tested in enzyme-linked immunosorbent assay (ELISA) for IgG binding in sera from 37 selected NPC patients who had IgG antibodies to the native ZEBRA protein. The most immunogenic epitope was peptide 1 at the amino-terminal end with 36 of the sera reactive against it. Further analysis of peptide 1, using the multipin peptide-scanning technique, defined a 10-amino-acid sequence FTPDPYQVPF, which was strongly bound by IgG. Two other regions of ZEBRA were also identified as immunodominant IgG epitopes, namely peptide 11 (amino acids 82-103) and peptide 19/20 (amino acids 146-175) with 8-13 of the NPC sera reactive against the peptides. The number of peptides reactive with individual NPC serum varies from 1 to 6 or more and there is some correlation between a greater number of peptide (at least 4) bound and a higher (at least 1:40) titre of serum IgA to viral capsid antigen. The immunodominant ZEBRA peptide 1 could be utilised in IgG ELISA for the detection of NPC.

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