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Fulltext Combined protein- and nucleic acid-level effects of rs1143679 (R77H), a lupus-predisposing variant within ITGAM

Maiti AK, Kim-Howard X, Motghare P, Pradhan V, Chua KH, Sun C, et al.

Hum Mol Genet, 2014 Aug 1;23(15):4161-76.
PMID: 24608226 DOI: 10.1093/hmg/ddu106

Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10(-90), odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele ('A') relative to the non-risk allele ('G'), in a dose-dependent fashion: ('AA' < 'AG' < 'GG'). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the 'A' transcript than 'G' transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE.

Matched MeSH terms: Antigens, Nuclear/genetics; Antigens, Nuclear/metabolism; Antigens, CD11b/genetics*; Antigens, CD11b/metabolism
Fulltext Susceptible and protective HLA class 1 alleles against dengue fever and dengue hemorrhagic fever patients in a Malaysian population

Appanna R, Ponnampalavanar S, Lum Chai See L, Sekaran SD

PLoS One, 2010;5(9).
PMID: 20927388 DOI: 10.1371/journal.pone.0013029

The human leukocyte antigen alleles have been implicated as probable genetic markers in predicting the susceptibility and/or protection to severe manifestations of dengue virus (DENV) infection. In this present study, we aimed to investigate for the first time, the genotype variants of HLA Class 1(-A and -B) of DENV infected patients against healthy individuals in Malaysia.

Matched MeSH terms: HLA-A Antigens/genetics*; HLA-A Antigens/immunology; HLA-B Antigens/genetics*; HLA-B Antigens/immunology
Cord blood CD34+ cells cultured with FLT3L, stem cell factor, interleukin-6, and IL-3 produce CD11c+CD1a-/c- myeloid dendritic cells

Fadilah SA, Vuckovic S, Khalil D, Hart DN

Stem Cells Dev, 2007 Oct;16(5):849-55.
PMID: 17999605

Methods that allow expansion of myeloid dendritic cells (MDCs) from CD34(+) cells are potentially important for boosting anti-leukemic responses after cord blood (CB) hematopoietic stem cell transplantation (HSCT). We showed that the combination of early-acting cytokines FLT3-ligand (FL), stem cell factor (SCF), interleukin (IL)-3, and IL-6 supported the generation of CD11c(+)CD16() CD1a()/c() MDCs from CB CD34(+) cells or CB myeloid precursors. Early-acting cytokine-derived MDCs were maintained within the myeloid CD33(+)CD14()CD15() precursors with a mean of 4 x 10(6) cells generated from 1-4 x 10(4) CB CD34(+) cells or myeloid precursors after 2 weeks. After 8-12 days of culture the MDCs expressed higher levels of HLA-DR antigen but lower levels of CD40 and CD86 antigen, compared to adult blood MDCs. At this stage of differentiation, the early-acting cytokine-derived MDCs had acquired the ability to induce greater allogeneic T cell proliferation than monocytes or granulocytes derived from same culture. Early-acting cytokine-derived MDCs exposed to the cytokine cocktail (CC) comprising IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and prostaglandin E (PGE)-2, upregulated the surface co-stimulatory molecules CD40 and CD86 and enhanced allogeneic T cell proliferation, as is characteristic of MDCs maturation. The reliable production of MDCs from CB CD34(+) cells provides a novel way to study their lineage commitment pathway(s) and also a potential means of enriching CB with MDCs to improve prospects for DC immunotherapy following CB HSCT.

Matched MeSH terms: Antigens, CD1/metabolism; Antigens, CD34/metabolism*; Antigens, CD11c/metabolism
The role of CD30, CD40 and CD95 in the regulation of proliferation and apoptosis in classical Hodgkin's lymphoma

Kim LH, Eow GI, Peh SC, Poppema S

Pathology, 2003 Oct;35(5):428-35.
PMID: 14555388

AIMS: CD30, CD40 and CD95 are members of the tumour necrosis factor receptor superfamily. Ligation to their respective ligands (CD30L, CD40L, CD95L) will generate a diverse set of signalling cascades. We aim to study the expression pattern of CD30, CD40 and CD95 in classical Hodgkin's lymphoma (cHL) and to correlate the expressions with proliferation and apoptosis in the Hodgkin/Reed-Sternberg (H/RS) cells of cHL with or without associated Epstein-Barr virus (EBV) infection.
METHODS: A total of 66 cHL cases were retrieved from the archives. Expressions of CD30, CD40, CD95 and proliferation by Ki-67 expression were detected with an immunohistochemical staining method. Apoptosis index was assessed by in situ TUNEL staining technique on 30 randomly selected cases and the presence of EBV was determined by EBER in situ hybridisation.
RESULTS: Expression of CD30, CD40 and CD95 in the H/RS cells was observed in a high proportion of the cases (100, 93.9, 90.5%, respectively). There was no significant association or correlation of the expression of these molecules with the presence of EBV. Expression of CD40 was associated with expression of the proliferation marker Ki-67 (P=0.044), whereas strong (intermediate and high) expression of CD30 showed a significant correlation with proliferation in the EBV-negative cases only (P=0.025). No correlation was observed for the expression of CD30 and CD40 with apoptosis of the H/RS cells. The childhood cases showed weaker CD95 expression in the H/RS cells than the adult cases, and the expression of CD95 was weaker than that of CD40 in the childhood group.
CONCLUSIONS: Our results showed that CD30, CD40 and CD95 are highly expressed in the H/RS cells of the majority of cases of cHL. The expression patterns seem to be independent of EBV and do not correlate with apoptosis of the H/RS cells.

Matched MeSH terms: Antigens, CD30/metabolism*; Antigens, CD40/metabolism*; Antigens, CD95/metabolism*
Generation of mesenchymal stem cell from human umbilical cord tissue using a combination enzymatic and mechanical disassociation method

Tong CK, Vellasamy S, Tan BC, Abdullah M, Vidyadaran S, Seow HF, et al.

Cell Biol Int, 2011 Mar;35(3):221-6.
PMID: 20946106 DOI: 10.1042/CBI20100326

MSCs (mesenchymal stem cells) promise a great potential for regenerative medicine due to their unique properties of self-renewal, high plasticity, modulation of immune response and the flexibility for genetic modification. Therefore, the increasing demand for cellular therapy necessitates a larger-scale production of MSC; however, the technical and ethical issues had put a halt on it. To date, studies have shown that MSC could be derived from human UC (umbilical cord), which is once considered as clinical waste. We have compared the two conventional methods which are classic enzymatic digestion and explant method with our newly tailored enzymatic-mechanical disassociation method to generate UC-MSC. The generated UC-MSCs from the methods above were characterized based on their immunophenotyping, early embryonic transcription factors expression and mesodermal differentiation ability. Our results show that enzymatic-mechanical disassociation method increase the initial nucleated cell yield greatly (approximately 160-fold) and maximized the successful rate of UC-MSC generation. Enzymatic-mechanical disassociation-derived UC-MSC exhibited fibroblastic morphology and surface markers expression of CD105, CD73, CD29, CD90 and MHC class I. Furthermore, these cells constitutively express early embryonic transcription factors (Nanog, Oct-4, Sox-2 and Rex-1), as confirmed by RT-PCR, indicating their multipotency and high self-renewal capacity. They are also capable of differentiating into osteoblasts and adipocytes when given an appropriate induction. The present study demonstrates a new and efficient approach in generating MSC from UC, hence serving as ideal alternative source of mesenchymal stem cell for clinical and research use.

Matched MeSH terms: Antigens, CD/metabolism; Antigens, Thy-1/metabolism; Antigens, CD29/metabolism
Fulltext Selection of phage-displayed human antibody fragments specific for CD1b presenting the Mycobacterium tuberculosis glycolipid Ac2SGL

Camacho F, Sarmiento ME, Reyes F, Kim L, Huggett J, Lepore M, et al.

Int J Mycobacteriol, 2016 06;5(2):120-7.
PMID: 27242221 DOI: 10.1016/j.ijmyco.2015.12.002

OBJECTIVE/BACKGROUND: The development of new tools capable of targeting Mycobacterium tuberculosis (Mtb)-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system (Mtb lipid-CD1b complexes). Because of the lack of CD1b polymorphism, specific Mtb lipid-CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-α'-d-trehalose (Ac2SGL) is specific for Mtb, and is not present in other mycobacterial species. The CD1b-Ac2SGL complexes are expressed on the surface of human cells infected with Mtb. The aim of this study was to generate ligands capable of binding these CD1b-Ac2SGL complexes.
METHODS: A synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b-Ac2SGL using CD1b-transfected THP-1 cells loaded with Ac2SGL.
RESULTS: One clone, D11-a single, light-variable domain (kappa) antibody (dAbκ11)-showed high relative binding to the Ac2SGL-CD1b complex.
CONCLUSION: A ligand recognizing the Ac2SGL-CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications.

Matched MeSH terms: Antigens, CD1/genetics; Antigens, CD1/immunology*
Brain endothelial cells increase the proliferation of Plasmodium falciparum through production of soluble factors

Khaw LT, Ball HJ, Mitchell AJ, Grau GE, Stocker R, Golenser J, et al.

Exp Parasitol, 2014 Oct;145:34-41.
PMID: 25045850 DOI: 10.1016/j.exppara.2014.07.002

We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3 kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.

Matched MeSH terms: Antigens, Protozoan/analysis; Antigens, Protozoan/metabolism
Fulltext Lateral flow test using Echinococcus granulosus native antigen B and comparison of IgG and IgG4 dipsticks for detection of human cystic echinococcosis

Khalilpour A, Sadjjadi SM, Moghadam ZK, Yunus MH, Zakaria ND, Osman S, et al.

Am J Trop Med Hyg, 2014 Nov;91(5):994-9.
PMID: 25200268 DOI: 10.4269/ajtmh.14-0170

Cystic echinococcosis (CE) caused by infection with Echinococcus granulosus is of major concern for humans in many parts of the world. Antigen B was prepared from E. granulosus hydatid fluid, and Western blots confirmed eight batches showing a band corresponding to the 8-/12-kDa subunit with positive serum and no low-molecular mass band (< 15 kDa) with negative serum. The batches were pooled and used to prepare lateral flow immunoglobulin G4 (IgG4) and IgG dipsticks. Diagnostic sensitivity was determined using serum samples from 21 hydatidosis patients, and diagnostic specificity was established using sera from 17 individuals infected with other parasites and 15 healthy people. IgG4 dipstick had a diagnostic sensitivity of 95% (20 of 21) and a specificity of 100% (32 of 32). The IgG dipstick had a sensitivity of 100% (21 of 21) and a specificity of 87.5% (28 of 32). Thus, both IgG and IgG4 dipsticks had high sensitivities, but IgG4 had greater specificity for the diagnosis of human CE.

Matched MeSH terms: Antigens, Helminth/blood; Antigens, Helminth/immunology
Fulltext Cost-effectiveness analysis of HLA-B*5801 testing in preventing allopurinol-induced SJS/TEN in Thai population

Saokaew S, Tassaneeyakul W, Maenthaisong R, Chaiyakunapruk N

PLoS One, 2014;9(4):e94294.
PMID: 24732692 DOI: 10.1371/journal.pone.0094294

BACKGROUND: Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN), caused by allopurinol therapy, are strongly associated with the human leukocyte antigen (HLA), HLA-B*5801. Identification of HLA-B*5801 genotype before prescribing allopurinol offers the possibility of avoiding allopurinol-induced SJS/TEN. As there is a paucity of evidence about economic value of such testing, this study aims to determine the cost-effectiveness of HLA-B*5801 testing compared with usual care (no genetic testing) before allopurinol administration in Thailand.
METHODS AND FINDING: A decision analytical and Markov model was used to estimate life time costs and outcomes represented as quality adjusted life years (QALYs) gained. The model was populated with relevant information of the association between gene and allopurinol-induced SJS/TEN, test characteristics, costs, and epidemiologic data for Thailand from a societal perspective. Input data were obtained from the literature and a retrospective database analysis. The results were expressed as incremental cost per QALY gained. A base-case analysis was performed for patients at age 30. A series of sensitivity analyses including scenario, one-way, and probabilistic sensitivity analyses were constructed to explore the robustness of the findings. Based on a hypothetical cohort of 1,000 patients, the incremental total cost was 923,919 THB (USD 29,804) and incremental QALY was 5.89 with an ICER of 156,937.04 THB (USD 5,062) per QALY gained. The cost of gout management, incidence of SJS/TEN, case fatality rate of SJS/TEN, and cost of genetic testing are considered very influential parameters on the cost-effectiveness value of HLA-B*5801 testing.
CONCLUSIONS: The genetic testing for HLA-B*5801 before allopurinol administration is considered a highly potential cost-effective intervention in Thailand. The findings are sensitive to a number of factors. In addition to cost-effectiveness findings, consideration of other factors including ethical, legal, and social implications is needed for an informed policy decision making.

Matched MeSH terms: HLA-B Antigens/economics*; HLA-B Antigens/genetics*
Current aspects in immunosensors

Gopinath SC, Tang TH, Citartan M, Chen Y, Lakshmipriya T

Biosens Bioelectron, 2014 Jul 15;57:292-302.
PMID: 24607580 DOI: 10.1016/j.bios.2014.02.029

Sensing applications can be used to report biomolecular interactions in order to elucidate the functions of molecules. The use of an analyte and a ligand is a common set-up in sensor development. For several decades, antibodies have been considered to be potential analytes or ligands for development of so-called "immunosensors." In an immunosensor, formation of the complex between antibody and antigen transduces the signal, which is measurable in various ways (e.g., both labeled and label-free based detection). Success of an immunosensor depends on various factors, including surface functionalization, antibody orientation, density of the antibody on the sensor platform, and configuration of the immunosensor. Careful optimization of these factors can generate clear-cut results for any immunosensor. Herein, current aspects, involved in the generated immunosensors, are discussed.

Matched MeSH terms: Antigens/analysis*; Antigens/immunology
Identification of Toxoplasma gondii in-vivo induced antigens by cDNA library immunoscreening with chronic toxoplasmosis sera

Amerizadeh A, Idris ZM, Khoo BY, Kotresha D, Yunus MH, Karim IZ, et al.

Microb Pathog, 2013 Jan;54:60-6.
PMID: 23044055 DOI: 10.1016/j.micpath.2012.09.006

Toxoplasmosis is an infection caused by the parasite Toxoplasma gondii. Chronically-infected individuals with a compromised immune system are at risk for reactivation of the disease. In-vivo induced antigen technology (IVIAT) is a promising method for the identification of antigens expressed in-vivo. The aim of the present study was to apply IVIAT to identify antigens which are expressed in-vivo during T. gondii infection using sera from individuals with chronic toxoplasmosis. Forty serum samples were pooled, pre-adsorped against three different preparations of antigens, from each in-vitro grown T. gondii and Escherichia coli XLBlue MRF', and then used to screen a T. gondii cDNA expression library. Sequencing of DNA inserts from positive clones showed eight open reading frames with high homology to T. gondii genes. Expression analysis using quantitative real-time PCR showed that SAG1-related sequence 3 (SRS3) and two hypothetical genes were up-regulated in-vivo relative to their expression levels in-vitro. These three proteins also showed high sensitivity and specificity when tested with individual serum samples. Five other proteins namely M16 domain peptidase, microneme protein, elongation factor 1-alpha, pre-mRNA-splicing factor and small nuclear ribonucleoprotein F had lower RNA expression in-vivo as compared to in-vitro. SRS3 and the two hypothetical proteins warrant further investigation into their roles in the pathogenesis of toxoplasmosis.

Matched MeSH terms: Antigens, Protozoan/genetics; Antigens, Protozoan/immunology*
Effects of symptomatic and asymptomatic isolates of Blastocystis hominis on colorectal cancer cell line, HCT116

Chan KH, Chandramathi S, Suresh K, Chua KH, Kuppusamy UR

Parasitol Res, 2012 Jun;110(6):2475-80.
PMID: 22278727 DOI: 10.1007/s00436-011-2788-3

The pathogenesis of Blastocystis hominis in human hosts has always been a matter of debate as it is present in both symptomatic and asymptomatic individuals. A recent report showed that B. hominis isolated from an asymptomatic individual could facilitate the proliferation and growth of existing cancer cells while having the potential to downregulate the host immune response. The present study investigated the differences between the effects of symptomatic and asymptomatic derived solubilized antigen of B. hominis (Blasto-Ag) on the cell viability and proliferation of colorectal cancer cells. Besides that, the gene expression of cytokine and nuclear transcriptional factors in response to the symptomatic and asymptomatic B. hominis antigen in HCT116 was also compared. In the current study, an increase in cell proliferation was observed in HCT116 cells which led to the speculation that B. hominis infection could facilitate the growth of colorectal cancer cells. In addition, a more significant upregulation of Th2 cytokines observed in HCT116 may lead to the postulation that symptomatic Blasto-Ag may have the potential in weakening the cellular immune response, allowing the progression of existing tumor cells. The upregulation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) was observed in HCT116 exposed to symptomatic Blasto-Ag, while asymptomatic Blasto-Ag exhibited an insignificant effect on NF-κB gene expression in HCT116. HCT116 cells exposed to symptomatic and asymptomatic Blasto-Ag caused a significant upregulation of CTSB which lead to the postulation that the Blasto-Ag may enhance the invasive and metastasis properties of colorectal cancer. In conclusion, antigen isolated from a symptomatic individual is more pathogenic as compared to asymptomatic isolates as it caused a more extensive inflammatory reaction as well as more enhanced proliferation of cancer cells.

Matched MeSH terms: Antigens, Protozoan/isolation & purification; Antigens, Protozoan/metabolism*
Fulltext Inhibition of dengue virus entry and multiplication into monocytes using RNA interference

Alhoot MA, Wang SM, Sekaran SD

PLoS Negl Trop Dis, 2011 Nov;5(11):e1410.
PMID: 22140591 DOI: 10.1371/journal.pntd.0001410

Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes.

Matched MeSH terms: Antigens, CD14/genetics; Antigens, CD14/metabolism*
Direct purification of recombinant hepatitis B core antigen from two different pre-conditioned unclarified Escherichia coli feedstocks via expanded bed adsorption chromatography

Ng MY, Tan WS, Abdullah N, Ling TC, Tey BT

J Chromatogr A, 2007 Nov 16;1172(1):47-56.
PMID: 17945242

Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.

Matched MeSH terms: Hepatitis B Core Antigens/isolation & purification*; Hepatitis B Core Antigens/chemistry
Allelic dimorphism in the merozoite surface protein-3alpha in Korean isolates of Plasmodium vivax

Han ET, Song TE, Park JH, Shin EH, Guk SM, Kim TY, et al.

Am J Trop Med Hyg, 2004 Dec;71(6):745-9.
PMID: 15642964

To study the genetic diversity of re-emerging Plasmodium vivax in the Republic of Korea, nucleotide sequence variations at the merozoite surface protein-3alpha (PvMSP-3alpha) locus were analyzed using 24 re-emerging isolates and 4 isolates from imported cases. Compared with the well known Belem strain (Brazil), a large number of amino acid substitutions, deletions, and insertions were found at the locus of the isolates examined. The Korean isolates were divided into two allelic types; type I (15 isolates), similar to the Belem strain, and type II (9), similar to the Chess strain (New Guinea). Isolates from imported cases were classified into three types; type III (1 from Malaysia), similar to type B from western Thailand, type IV (1 each from Indonesia and India), and type V (1 from Pakistan), both being new types. Our results have shown that the MSP-3alpha locus of re-emerging Korean P. vivax is dimorphic with two allelic types coexisting in the endemic area.

Matched MeSH terms: Antigens, Protozoan/genetics*; Antigens, Protozoan/chemistry
Solution structure and in silico binding of a cyclic peptide with hepatitis B surface antigen

Muhamad A, Ho KL, Rahman MB, Uhrín D, Tan WS

Chem Biol Drug Des, 2013 Jun;81(6):784-94.
PMID: 23405984 DOI: 10.1111/cbdd.12120

A specific ligand targeting the immunodominant region of hepatitis B virus is desired in neutralizing the infectivity of the virus. In a previous study, a disulfide constrained cyclic peptide cyclo S(1) ,S(9) Cys-Glu-Thr-Gly-Ala-Lys-Pro-His-Cys (S(1) , S(9) -cyclo-CETGAKPHC) was isolated from a phage displayed cyclic peptide library using an affinity selection method against hepatitis B surface antigen. The cyclic peptide binds tightly to hepatitis B surface antigen with a relative dissociation constant (KD (rel) ) of 2.9 nm. The binding site of the peptide was located at the immunodominant region on hepatitis B surface antigen. Consequently, this study was aimed to elucidate the structure of the cyclic peptide and its interaction with hepatitis B surface antigen in silico. The solution structure of this cyclic peptide was solved using (1) H, (13) C, and (15) N NMR spectroscopy and molecular dynamics simulations with NMR-derived distance and torsion angle restraints. The cyclic peptide adopted two distinct conformations due to the isomerization of the Pro residue with one structured region in the ETGA sequence. Docking studies of the peptide ensemble with a model structure of hepatitis B surface antigen revealed that the cyclic peptide can potentially be developed as a therapeutic drug that inhibits the virus-host interactions.

Matched MeSH terms: Hepatitis B Surface Antigens/metabolism*; Hepatitis B Surface Antigens/chemistry
Genotyping of hepatitis B virus in Malaysia based on the nucleotide sequence of preS and S genes

Ong HT, Duraisamy G, Kee Peng N, Wen Siang T, Seow HF

Microbes Infect., 2005 Mar;7(3):494-500.
PMID: 15792534

Hepatitis B virus (HBV) has been classified into eight genotypes, designated A-H. These genotypes are known to have distinct geographic distributions. The clinical importance of genotype-related differences in the pathogenicity of HBV has been revealed recently. In Malaysia, the current distribution of HBV remains unclear. The aim of this study was to determine the genotypes and subtypes of HBV by using PCR, followed by DNA sequencing, as well as to analyse the mutations in the immunodominant region of preS and S proteins. The S gene sequence was determined from HBV DNA of four apparently healthy blood donors' sera and three sera from asymptomatic chronic hepatitis B carriers. Of this batch of sera, the preS gene sequence was obtained from HBV DNA from three out of the four blood donors and two out of the three chronic carriers. Due to insufficient sera, we had to resort to using sera from another blood donor to make up for the sixth DNA sequence of the preS gene. Based on the comparative analysis of the preS sequences with the reported sequences in the GenBank database, HBV DNA from two normal carriers was classified as genotype C. Genotype B was assigned to HBV from one blood donor and two hepatitis B chronic carriers, whereas HBV of one chronic carrier was of genotype D. Based on the S gene sequences, HBV from three blood donors was of genotype C, that of one blood donor and one chronic carrier was of genotype B, and the remaining, of genotype D. In the five cases where both preS and S gene sequences were determined, the genotypes assigned based on either the preS or S gene sequences were in concordance. The nature of the deduced amino acid (aa) sequences at positions 125, 127, 134, 143, 159, 161 and 168 of the S gene enabled the classification of these sequences into subtypes, namely, adrq+, adw2 and ayw2. The clustering of our DNA sequences into genotype groups corresponded to their respective subtype, that is, adw2 in genotype B, adrq in genotype C and ayw in genotype D. Analysis of the point mutations revealed that five of the sequences contained aa substitutions at immunodominant epitopes involved in B or/and T cell recognition. In conclusion, despite the low numbers of samples studied, due to budget constraints, these data are still worthwhile reporting, as it is important for the control of HBV infections. In addition, the genotype and mutational data obtained in this study may be useful for designing new treatment regimes for HBV patients.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics*; Hepatitis B Surface Antigens/chemistry
Fulltext Crystal structure of Plasmodium knowlesi apical membrane antigen 1 and its complex with an invasion-inhibitory monoclonal antibody

Vulliez-Le Normand B, Faber BW, Saul FA, van der Eijk M, Thomas AW, Singh B, et al.

PLoS One, 2015;10(4):e0123567.
PMID: 25886591 DOI: 10.1371/journal.pone.0123567

The malaria parasite Plasmodium knowlesi, previously associated only with infection of macaques, is now known to infect humans as well and has become a significant public health problem in Southeast Asia. This species should therefore be targeted in vaccine and therapeutic strategies against human malaria. Apical Membrane Antigen 1 (AMA1), which plays a role in Plasmodium merozoite invasion of the erythrocyte, is currently being pursued in human vaccine trials against P. falciparum. Recent vaccine trials in macaques using the P. knowlesi orthologue PkAMA1 have shown that it protects against infection by this parasite species and thus should be developed for human vaccination as well. Here, we present the crystal structure of Domains 1 and 2 of the PkAMA1 ectodomain, and of its complex with the invasion-inhibitory monoclonal antibody R31C2. The Domain 2 (D2) loop, which is displaced upon binding the Rhoptry Neck Protein 2 (RON2) receptor, makes significant contacts with the antibody. R31C2 inhibits binding of the Rhoptry Neck Protein 2 (RON2) receptor by steric blocking of the hydrophobic groove and by preventing the displacement of the D2 loop which is essential for exposing the complete binding site on AMA1. R31C2 recognizes a non-polymorphic epitope and should thus be cross-strain reactive. PkAMA1 is much less polymorphic than the P. falciparum and P. vivax orthologues. Unlike these two latter species, there are no polymorphic sites close to the RON2-binding site of PkAMA1, suggesting that P. knowlesi has not developed a mechanism of immune escape from the host's humoral response to AMA1.

Matched MeSH terms: Antigens, Protozoan/immunology; Antigens, Protozoan/chemistry*
Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei

Chenthamarakshan V, Vadivelu J, Puthucheary SD

Diagn Microbiol Infect Dis, 2001 Jan;39(1):1-7.
PMID: 11173184

IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.

Matched MeSH terms: Antigens, Bacterial/analysis; Antigens, Bacterial/immunology*
Detection of Angiostrongylus malaysiensis circulating antigen using monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA)

Ambu S, Rain AN, Mak JW, Maslah D, Maidah S

Southeast Asian J. Trop. Med. Public Health, 1997;28 Suppl 1:143-7.
PMID: 9656366

Three MAbs 1C4.2D8, 1C4.2C4 and 1C4.1F5 were produced using sonicated adult worm antigens of Angiostrongylus malaysiensis and they were found to be secreters of IgG1. The MAbs 1C4.2C4 and 1C4.2D8 were found to react with antigens of A. malaysiensis and cross-react with the closely related A. cantonensis but not with other helminths. A total of 108 human sera collected from Orang Asli (aborigenes) from Grik, in the State of Perak were tested for A. malaysiensis infection using the MAb-ELISA. MAb 1C4.1F5 and 25 (23%) were positive. Twenty of these positive samples were tested with the MAb 1C4.2D8 and none was found to be positive.

Matched MeSH terms: Antigens, Helminth/blood*; Antigens, Helminth/immunology

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