Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.
Hybridoma clone C3A8, which is a fusion product between splenic lymphocytes of Balb/c mice immunized with MCF7 breast carcinoma cells and SP2/0 myelomas, was produced and characterized. A stable clone that secreted IgM monoclonal antibody (MAb) with kappa light chain was obtained through limiting dilutions. Cell-ELISA screening, flow cytometry analysis, and immunofluorescence staining revealed that the MAb C3A8 had bound specifically and strongly to MCF7 and HT29 but cross reacted weakly or not on HeLa cell line. The MAb C3A8 reacted positively with paraffin-embedded tissues of human breast and colon cancers but there were no positive reactions on normal tissues. Western blot analysis showed the MAb recognized a 55 kDa protein, which was present in the extract of MCF7 and HT29 cell lines. Our results demonstrated that MAb C3A8 could be used for basic and clinical research of breast and colon cancers.
Lichen planus, a chronic autoimmune, mucocutaneous disease affects the oral mucosa (oral lichen planus or OLP) besides the skin, genital mucosa, scalp and nails. An immune mediated pathogenesis is recognized in lichen planus although the exact etiology is unknown. The disease most commonly affects middle-aged females. Oral lichenoid reactions (OLR) which are considered variants of OLP, may be regarded as a disease by itself or as an exacerbation of an existing OLP, by the presence of medication (lichenoid drug reactions) or dental materials (contact hypersensitivity). OLP usually presents as white striations (Wickham's striae), white papules, white plaque, erythema, erosions or blisters. Diagnosis of OLP is established either by clinical examination only or by clinical examination with histopathologic confirmation. Direct immunofluorescence examination is only used as an adjunct to the above method of diagnosis and to rule out specific autoimmune diseases such as pemphigus and pemphigoid. Histopathologic features of OLP and OLR are similar with suggestions of certain discriminatory features by some authors. Topical corticosteroids are the treatment of choice for OLP although several other medications have been studied including retinoids, tacrolimus, cyclosporine and photodynamic therapy. Certain OLP undergo malignant transformation and the exact incidence and mechanisms are still controversial. In this paper, etiopathogenesis, diagnosis, management and malignant transformation of OLP and OLR have been reviewed.
Matched MeSH terms: Fluorescent Antibody Technique, Direct
Systemic Lupus Erythematosus (SLE) is a disease with multiorgan involvement and multiple autoantibody production including antineutrophil cytoplasmic antibodies (ANCA). Despite its reported prevalence in more than one third of SLE patients, the role of ANCA in the pathogenesis or otherwise in SLE remains unresolved. 131 SLE patients had been previously studied for various serologic parameters of disease activity. Their cumulative organ involvement in the course of their disease had also been determined and the Lupus Activity Index (LAI) calculated. Their stored sera were then screened for the presence of ANCA by two methods viz Indirect immunofluorescence (IIF) and also enzyme-linked immunosorbent assay (ELISA). ANCA was present in 24.8% of these SLE patients. The atypical ANCA pattern was predominant and accounted for an overall of 20.6%. Anti-MPO and anti-PR3 were detected in 1.5% of patients respectively. No association was found between ANCA positivity and disease activity. There was also no association of ANCA with specific organ involvement. Despite the high prevalence of ANCA especially the atypical variant in SLE, they probably represent only one of the wide repertoire of autoantibodies found in this disease. Routine testing for ANCA in lupus patients is therefore not recommended.
The traditional application of the sclerotium of Lignosus rhinocerotis (tiger's milk mushroom) by the indigenous folks as tonic and remedy to treat a variety of ailments has been documented in Malaysia. Indigenous communities claimed to have consumed the decoction to boost their alertness during hunting. Mental alertness is believed to be related to neuronal health and neuroactivity. In the present study, the cell viability and neuritogenic effects of L. rhinocerotis sclerotium hot aqueous and ethanolic extracts, and crude polysaccharides on rat pheochromocytoma (PC-12) cells were studied. Interestingly, the hot aqueous extract exhibited neuritogenic activity comparable to NGF in PC-12 cells. However, the extracts and crude polysaccharides stimulated neuritogenesis without stimulating the production of NGF in PC-12 cells. The involvements of the TrkA receptor and MEK/ERK1/2 pathway in hot aqueous extract-stimulated neuritogenesis were examined by Trk (K252a) and MEK/ERK1/2 (U0126 and PD98059) inhibitors. There was no significant difference in protein expression in NGF- and hot aqueous extract-treated cells for both total and phosphorylated p44/42 MAPK. The neuritogenic activity in PC-12 cells stimulated by hot aqueous and ethanolic extracts, and crude polysaccharides of L. rhinocerotis sclerotium mimicking NGF activity via the MEK/ERK1/2 signaling pathway is reported for the first time.
Exanthem subitum (ES) is a common childhood exanthematous disease. In a recent study of ES due to human herpesvirus 6 (HHV 6), we isolated human herpesvirus 7 (HHV 7) from the peripheral blood mononuclear cells (PBMC) of a seven month-old infant with typical symptoms of ES. The identity of the virus was confirmed by indirect immunofluorescence using HHV 7 specific monoclonal antibody and by amplification of the HHV 7 specific genomic sequences using the polymerase chain reaction (PCR). Paired serum samples from the infant showed serological conversion to the isolated virus. The clinical manifestations of ES in this infant appeared to be milder than the classical ES due to HHV 6.
A study was undertaken to evaluate the ability of flow cytometric analysis of intracellular myeloperoxidase (MPO) in differentiating populations of lymphocytes (L), monocytes (M) and granulocytes (G), by means of lysed whole blood method. Anticoagulated blood from 23 normal individuals was lysed with FACS lysing solution and permeabilized with FACS permeabilizing solution before subjected to direct immunofluorescence staining. The geometric means of the fluorescence intensity were measured using FACSCalibur flow cytometer (Becton Dickinson). Populations of L, M and G were gated based on their light scatter characteristics and expression of CD14 and CD45. Then, the fluorescence intensity of MPO expression was studied in these individual cell populations. The results showed that fluorescence intensity of MPO was the strongest in G and weakest in L, whereas M showed intermediate fluorescence intensity. Our findings reveal that discrimination of these three cell types is achievable based upon the sole expression of intracellular MPO.
Matched MeSH terms: Fluorescent Antibody Technique, Direct
Zingiberaceae rhizomes commonly used in the Malaysian traditional medicine were screened for anti-tumour promoter activity using the short-term assay of inhibition of 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) in Raji cells. The inhibition of TPA-induced EBV-EA was detected using the indirect immunofluorescence assay (IFA) and Western blot technique. The indirect IFA detected the expression/inhibition of EBV-EA-D (diffused EA antigen), whereas the Western blot technique detected the expression/inhibition of both EBV-EA-D and EA-R (restricted EA antigen). Seven rhizomes were found to possess inhibitory activity towards EBV activation, induced by TPA; they are: Curcuma domestica, C. xanthorrhiza, Kaempferia galanga, Zingiber cassumunar, Z. officinale, Z. officinale (red variety), and Z. zerumbet. A cytotoxicity assay was carried out to determine the toxicity of the Zingiberaceae rhizome extracts. The rhizome extracts that exhibited EBV activation inhibitory activity had no cytotoxicity effect in Raji cells. Therefore, the present study shows that several Zingiberaceae species used in Malaysian traditional medicine contain naturally occurring non-toxic compounds that inhibit the EBV activation, which, if further investigated, could contribute in the development of cancer prevention methods at the tumour-promoting stage.
A dot-immunobinding assay (DIBA) was compared with a direct fluorescent antibody technique (DFAT) for the detection of Rickettsia tsutsugamushi infection in Leptotrombidium fletcheri (Womersley & Heaslip). Laboratory colonies of infected and noninfected chiggers were examined. The relative proportions of positive, negative, and indeterminate results were significantly different between DIBA and DFAT for infected but not for noninfected chiggers. DIBA was more sensitive and had a better negative predictive value and a lower false negative percentage than DFAT. It was concluded that DIBA is a suitable alternative to DFAT for detecting scrub typhus infection in chiggers.
The occurrence of a case of human rabies in Peninsular Malaysia is reported. Despite the various control measures taken, sporadic cases of rabies have continued to occur in Peninsular Malaysia, especially in the northern states. Clinical awareness of the occurrence of rabies is therefore important and effective post-exposure prophylaxis should be instituted as soon as possible to prevent the possible occurrence of this dreaded disease.
A seroepidemiological study was carried out on Orang Asli (Aborigines) children who lead a semi-nomadic life in the deep jungles of Ulu Kelantan, Malaysia. Out of a total of about 190 children below 14 years, 143 were studied. Blood was collected from finger pricks on standard "strip type" filter papers for indirect fluorescent antibody (IFA) tests with Plasmodium falciparum antigen. A positive reaction at 1:10 dilution in infants and young children was considered positive and the reasons are given. The P. falciparum antibody prevalence rate was 84.6% compared to 81.8% spleen and 43.4% parasite rates. Both P. Falciparum and P. vivax were present in children. The age-specific patterns of antibody, spleen and parasite rates were those of a hyperendemic community. There was a positive correlation between antibody and spleen rates up to the age of 9 years. In older children, the antibody rates increased while the spleen and the parasite rates dropped.
In the 10-year period from October 1977 to July 1987, 149 cases of primary IgA nephropathy were histologically confirmed through renal biopsies in the Department of Pathology, University Hospital, Kuala Lumpur. The ages of these patients ranged from 5 to 72 years, with the majority in the 20-30 year age group. There was no sex preponderance. The ethnic distribution showed a significant predominance of Chinese with 107 (71.8%) Chinese, 24 (16.1%) Malays, 15 (10.1%) Indians and 3 others. A wide range of renal glomerular pathology was seen, the commonest being diffuse mesangioproliferative glomerulonephritis (59.1%). Focal proliferative glomerulonephritis (14.1%) followed by minimal change glomerulonephritis (10.7%) were next in order of frequency. Immunofluorescence studies consistently demonstrated heavy and predominant IgA deposition in the mesangium. Weak deposition of C3, IgG and IgM were also observed in various combinations.
Using an indirect immunofluorescence technique, sera from 113 cynomolgus monkeys (Macaca fascicularis), trapped in Peninsular Malaysia, were screened for the presence of antibody to six prototype strains of Rickettsia tsutsugamushi combined into three polyvalent groupings: I--Karp, TA716, and TA763; II--Gilliam; and III--TA678 and TH1817. Fifteen percent (17/113) of the monkeys had antibody titers greater than or equal to 1:50 to one or more of the antigenic groups. Although a titer greater than or equal to 1:150 is generally considered indicative or prior Rickettsia tsutsugamushi infection, we selected a less than 1:25 titer as a conservative standard to insure non-infected animals. Using this criterion, 62 (55%) of the 113 monkeys were accepted for use in scrub typhus studies. The high prevalence of antibody to scrub typhus in the semi-arboreal cynomolgus monkey is in marked contrast to the low prevalence reported in the strictly arboreal silvered leaf monkeys (Presbytis cristatus). The results of this study indicate that cynomolgus monkeys should be rigorously screened for evidence of prior infection before they are included in experimental scrub typhus studies.
Sera obtained from 3,472 persons in Malaysia, Thailand, Philippines and Indonesia were tested for the presence of antibody to adult T-cell leukemia-associated antigen by the gelatin particle agglutination test and indirect immunofluorescence. Among these, only two seropositives were identified. One was a 30-year-old male Malaysian of Indian origin. The other was a 42-year-old female Thai who resided in Bangkok. These results suggested that the infection of human T-lymphotropic virus type 1 might not be endemic in these countries.
One hundred and fourteen Rickettsia tsutsugamushi isolates, recovered from febrile patients in central Peninsular Malaysia, were antigenically analyzed by direct immunofluorescence using eight prototype strains. Twenty-nine antigenic types were detected. The TA763, TA716, Karp and TA686 strains were the most common and occurred singly or in combination with each other or other strains in 86% of the isolates.
Tumorigenesis involves a complex interplay between genetically modified cancer cells and their adjacent normal tissue, the stroma. We used an established breast cancer mouse model to investigate this inter-relationship. Conditional activation of Rho-associated protein kinase (ROCK) in a model of mammary tumorigenesis enhances tumor growth and progression by educating the stroma and enhancing the production and remodeling of the extracellular matrix. We used peptide matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify the proteomic changes occurring within tumors and their stroma in their regular spatial context. Peptides were ranked according to their ability to discriminate between the two groups, using a receiver operating characteristic tool. Peptides were identified by liquid chromatography tandem mass spectrometry, and protein expression was validated by quantitative immunofluorescence using an independent set of tumor samples. We have identified and validated four key proteins upregulated in ROCK-activated mammary tumors relative to those expressing kinase-dead ROCK, namely, collagen I, α-SMA, Rab14, and tubulin-β4. Rab14 and tubulin-β4 are expressed within tumor cells, whereas collagen I is localized within the stroma. α-SMA is predominantly localized within the stroma but is also expressed at higher levels in the epithelia of ROCK-activated tumors. High expression of COL1A, the gene encoding the pro-α 1 chain of collagen, correlates with cancer progression in two human breast cancer genomic data sets, and high expression of COL1A and ACTA2 (the gene encoding α-SMA) are associated with a low survival probability (COLIA, p = 0.00013; ACTA2, p = 0.0076) in estrogen receptor-negative breast cancer patients. To investigate whether ROCK-activated tumor cells cause stromal cancer-associated fibroblasts (CAFs) to upregulate expression of collagen I and α-SMA, we treated CAFs with medium conditioned by primary mammary tumor cells in which ROCK had been activated. This led to abundant production of both proteins in CAFs, clearly highlighting the inter-relationship between tumor cells and CAFs and identifying CAFs as the potential source of high levels of collagen 1 and α-SMA and associated enhancement of tissue stiffness. Our research emphasizes the capacity of MALDI-MSI to quantitatively assess tumor-stroma inter-relationships and to identify potential prognostic factors for cancer progression in human patients, using sophisticated mouse cancer models.
Introduction: Systemic lupus erythematosus (SLE) has a broad spectrum of clinical presentations. The diagnosis of SLE remains a challenge and largely depends on the presence of several serum autoantibodies including anti-nuclear antibody (ANA), anti-double-stranded DNA antibody (anti-dsDNA) and anti-Smith antibody (anti-Sm). ANA, a highly sensitive but not specific marker is used for SLE screening Anti-dsDNA and anti-Sm are SLE-specific biomarkers but has lower sensitivity of 80% and 30% for SLE, respectively. However, it is noted that there are SLE patients who are persistently negative for SLE-specific autoantibodies. Anti-dsDNA and anti-Sm were reported to be negative in up to 51.2% and 62.4% of SLE, respectively. This limitation can lead to misdiagnosis and halter proper treatment to SLE patients. Previous studies have suggested that cell membrane DNA (cmDNA) can act as a specific target for the autoantibodies in SLE patients. Autoantibodies towards cmDNA (anti-cmDNA) were reported to have promis-ing value as a reliable biomarker for SLE. In this study, we would like to determine the usefulness of anti-cmDNA in diagnosing SLE as compared to the standard SLE-specific autoantibodies. Methods: Serum samples from 83 SLE patients, 86 other connective tissue diseases and 61 healthy subjects were included in this study. The other connec-tive tissue diseases include samples from 10 Sjogren’s syndrome, 56 rheumatoid arthritis, 12 scleroderma and eight mixed connected tissues disease (MCTD) patients. All samples were analysed by indirect immunofluorescence (IIF) technique using Raji cells as substrate to detect the presence of anti-cmDNA. Anti-cmDNA was reported as positive if there was presence of a fluorescent ring, either continuous or punctate. Sera from SLE patients were also tested for anti-dsDNA and anti-Sm antibodies by using enzyme-immunoassays. Results: Anti-cmDNA positivity was highest in SLE (55.4%) than in other connective tissue diseases (9.3%) and healthy subjects (0%). Anti-cmDNA was 100% spe-cific at differentiating SLE from healthy subjects and 90.7% specific at differentiating SLE from other connective tissue diseases. There was no difference in the sensitivity (55.4%) of anti-cmDNA at differentiating SLE from both groups. Anti-cmDNA were present in 46 SLE samples negative for standard SLE-specific autoantibodies. It was detected in 11 (42.3%) of anti-dsDNA, 23 (63.9%) of anti-Sm and 8 (12.9%) of both anti-Sm and anti-dsDNA negative samples. Conclusion: The high specificity of anti-cmDNA detection using IIF method makes it an excellent diagnostic tool for SLE. Anti-cmDNA is potentially a very useful biomarker for SLE with negative anti-dsDNA or/and anti-Sm antibodies.
There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labeling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pretreatment with silver nanowires (AgNW) using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N+ = 70 amu), fragments of the sulfur-producing cystathionine-containing enzymes, and the Ag+ ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N+ amino acid fragment. Scanning electron microscopy imaging and energy-dispersive X-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur-producing proteins were detected. The presence of these sulfur-producing cystathionine-containing enzymes within the cells was confirmed by Western blots and confocal microscopy images of fluorescently labeled antibodies against the sulfur-producing enzymes. Label-free TOF-SIMS is very promising for the label-free identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in the future be used to identify which of these enzymes are most contributory.