Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P
An indirect immunoperoxidase test was compared with an indirect fluorescent antibody test and the Weil-Felix OXK test for serodiagnosis of scrub typhus by measuring the rickettsial antigen specific activity of IgG, IgM, and whole globulin. Acute and convalescent sera from 50 Rickettsia tsutsugamushi isolate-positive scrub typhus patients and from 45 febrile patients diagnosed as having diseases other than scrub typhus were tested. The receiver operating characteristic for each test showed that the indirect immunoperoxidase and indirect fluorescent antibody tests were more sensitive and specific than the Weil-Felix test using convalescent and acute as well as paired sera. The indirect immunoperoxidase test showed no cross-reactivity when R. tsutsugamushi antigen was tested against sera collected from patients living outside the scrub typhus-endemic area with diseases other than scrub typhus. The indirect immunoperoxidase and indirect fluorescent antibody tests were comparable in measured response to R. tsutsugamushi, R. typhi, and TT-118 (spotted fever group) antigen. Thus the indirect immunoperoxidase test represents a sensitive, specific, reproducible, and practical semiquantitative test for rickettsial disease diagnosis.
Background: Hepatitis B virus (HBV) infection has many faces. Precore and core promoter mutants resemble inactive carrier status. The identification of hepatitis B core antigen (HBcAg) in hepatocytes may have variable clinical significance. The present study was undertaken to detect HBcAg in chronic hepatitis B (CHB) patients and to assess the efficacy of detection system by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP).
Materials and methods: The study was done in 70 chronic HBV-infected patients. Out of 70 patients, eight (11.4%) were hepatitis B e antigen (HBeAg) positive and 62 (88.57%) were HBeAg negative. Hepatitis B core antigen was detected by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP) methods in liver tissue.
Results: All HBeAg positive patients expressed HBcAg by both IIF and IIP methods. Out of 62 patients with HBeAg-negative CHB, HBcAg was detected by IIF in 55 (88.7%) patients and by IIP in 51 (82.26%) patients. A positive relation among viral load and HBcAg detection was also found. This was more evident in the case of HBeAg negative patients and showed a positive relation with HBV DNA levels.
Conclusion: Hepatitis B core antigen can be detected using the IIF from formalin fixed paraffin block preparation and also by IIP method. This seems to reflect the magnitudes of HBV replication in CHB.
How to cite this article: Raihan R, Tabassum S, Al-Mahtab M, Nessa A, Jahan M, Kabir CMS, Kamal M, Aguilar JC. Hepatitis B Core Antigen in Hepatocytes of Chronic Hepatitis B: Comparison between Indirect Immunofluorescence and Immunoperoxidase Method. Euroasian J Hepato-Gastroenterol 2015;5(1):7-10.
N-cadherin is a calcium-sensitive cell adhesion molecule that plays an important role in the formation of the neural circuit and the development of the nervous system. In the present study, we investigated the function of N-cadherin in cell-cell connection in vitro with HEK293T cells, and in commissural axon projections in the developing chicken spinal cord using in ovo electroporation. Cell-cell connections increased with N-cadherin overexpression in HEK293T cells, while cell contacts disappeared after co-transfection with an N-cadherin-shRNA plasmid. The knockdown of N-cadherin caused the accumulation of β-catenin in the nucleus, supporting the notion that N-cadherin regulates β-catenin signaling in vitro. Furthermore, N-cadherin misexpression perturbed commissural axon projections in the spinal cord. The overexpression of N-cadherin reduced the number of axons that projected alongside the contralateral margin of the floor plate, and formed intermediate longitudinal commissural axons. In contrast, the knockdown of N-cadherin perturbed commissural axon projections significantly, affecting the projections alongside the contralateral margin of the floor plate, but did not affect intermediate longitudinal commissural axons. Taken together, these findings suggest that N-cadherin regulates commissural axon projections in the developing chicken spinal cord.
In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.
The sharp increase in incidence of dengue infection has necessitated the development of methods for the rapid diagnosis of this deadly disease. Here we report the design and development of a reliable, sensitive, and specific optical immunosensor for the detection of the dengue nonstructural protein 1 (NS1) biomarker in clinical samples obtained during early stages of infection. The present optical NS1 immunosensor comprises a biosensing surface consisting of specific monoclonal NS1 antibody for immunofluorescence-based NS1 antigen determination using fluorescein isothiocyanate (FITC) conjugated to IgG antibody. The linear range of the optical immunosensor was from 15-500ngmL-1, with coefficient of determination (R2) of 0.92, high reproducibility (the relative standard deviation obtained was 2%), good stability for 21days at 4°C, and low detection limit (LOD) at 15ngmL-1. Furthermore, the optical immunosensor was capable of detecting NS1 analytes in plasma specimens from patients infected with the dengue virus, with low cross-reaction with plasma specimens containing the Japanese encephalitis virus (JEV) and Zika virus. No studies have been performed on the reproducibility and cross-reactivity regarding NS1 specificity, which is thus a limitation for optical NS1 immunosensors. In contrast, the present study addressed these limitations carefully where these two important experiments were conducted to showcase the robustness of our newly developed optical-based fluorescence immunosensor, which can be practically used for direct NS1 determination in any untreated clinical sample.
INTRODUCTION: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection.
METHODOLOGY: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.
RESULTS: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays.
CONCLUSIONS: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.
Hepatocellular carcinoma (HCC) is among the ten most common cancers in Malaysian males. As cellular proliferation is an important feature of malignant transformation, we studied the proliferation pattern of normal and benign perineoplastic liver versus hepatocellular carcinoma in an attempt to further understand the tumour transformation process. 39 HCC (21 with accompanying and 18 without cirrhosis) histologically diagnosed at the Department of Pathology, University of Malaya Medical Centre between January 1992 and December 2003 were immunohistochemically studied using a monoclonal antibody to PCNA (Clone PC10: Dako). 20 livers from cases who had succumbed to traumatic injuries served as normal liver controls (NL). PCNA labeling index (PCNA-LI) was determined by counting the number of immunopositive cells in 1000 contiguous HCC, benign cirrhotic perineoplastic liver (BLC), benign perineoplastic non-cirrhotic (BLNC) and NL cells and conversion to a percentage. The PCNA-LI was also expressed as Ojanguren et al's grades. PCNA was expressed in 10% NL, 38.9% BLNC, 76.2% BLC and 71.8% HCC with BLNC, BLC and HCC showing significantly increased (p < 0.05) number of cases which expressed PCNA compared with NL. The number of BLC which expressed PCNA was also significantly increased compared with BLNC. PCNA-LI ranged from 0-2.0% (mean = 0.2%) in NL, 0-2.0% (mean = 0.3%) in BLNC, 0-3.6% (mean = 0.7%) in BLC and 0-53.8% (mean = 7.6%) in HCC with PCNA-LI significantly increased (p < 0.05) only in HCC compared with BLC, BLNC and NL. Accordingly, all NL, BLC and BLNC showed minimal (<5% cells being immunopositive) immunoreactivity on Ojanguren et al's grading system and only HCC demonstrated immunoreactivity which ranged up to grade 3 (75% of cells). From this study, there appears to be a generally increasing trend of proliferative activity from NL to BLNC to BLC and HCC. Nonetheless, BLNC and BLC, like NL, retained low PCNA-LI and only HCC had a significantly increased PCNA-LI compared with the benign categories. This is probably related to the malignant nature of HCC and may reflect the uncontrolled proliferation of the neoplastic hepatocytes.
Matched MeSH terms: Fluorescent Antibody Technique, Direct
Survivin is an inhibitor of apoptosis protein and regulates the cell cycle in the G2/M phase. Survivin is expressed during embryonic and fetal development, selectively over-expressed in common human cancers and completely down-regulated in normal adult tissue. This work was aimed at studying the expression of the survivin homologues and their subcellular distribution in fetal and normal adult tissues of rat. Survivin expression was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue sections of fetal and normal adult tissues of rat using the polyclonal serum SUR12A-CFI. This serum demonstrated intense positive survivin staining in adult kidney, ovary and oviduct, and a variable expression in different fetal organs, with particularly intense expression detected in the adrenal gland, liver, stomach, small intestine, colon, kidney and skin. In both fetal and adult tissues, the expression was predominantly cytoplasmic. It was concluded that survivin was abundantly and prominently expressed during fetal development in rat and that the polyclonal anti-human survivin antibody SUR12A-CFI is reactive with rat survivin.
In mid-1997, several children died in Sarawak, Malaysia, during an epidemic of enterovirus-71 (EV71) hand, foot, and mouth disease. The children who died had a febrile illness that rapidly progressed to cardiopulmonary failure and the cause was not satisfactorily resolved. We describe the isolation and identification of a subgenus B adenovirus from the children who died.
Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.
Two different preparations of monoclonal antibodies developed against respiratory viruses have been evaluated by the immunofluorescence antibody technique. The Chemicon monoclonal antibodies were found to be more efficient at picking up positive specimens with a high sensitivity and specificity than Imagen monoclonal antibodies. However, the overall concordance rate of the monoclonal antibodies was 92.3%-100%. Generally, when compared with cell culture isolation, the immunofluorescence antibody technique was found to be more sensitive. The high quality of the Chemicon monoclonal antibodies contribute to their value in providing definitive diagnosis, within a few hours of specimen collection, thus allowing early management of patients, their contacts and control of hospital infection.
It was reported that a 47kDa antigenic polypeptide of Plasmodium falciparum had been strongly presented by the sera from 1) imported Japanese malaria patients with severe symptoms and 2) symptomatic and parasitemic inhabitants in endemic areas in the Sudan, Malaysia and the Philippines. In the present study, we observed the reactivity of the sera from falciparum malaria patients who had been hospitalized in the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, and compared the antibody response against the 47kDa antigenic polypeptide according to the severity of the patients. It was observed that antibodies to this molecule were more commonly shared in sera from severer patients, although the IFAT titers against the whole P. falciparum parasite antigen were lower in the group, which suggested that this antibody against the 47kDa molecule was playing a specific role at a severe stage of the infection. Determination of the immunological features of the antigenic molecules of parasites by this type of sero-epidemiological study will provide a new assay system for evaluation of immune status of individuals in different severity and suggest a way of vaccine development.
A rapid diagnostic system for scrub typhus using nested polymerase chain reaction (PCR) was applied to clinical samples from Malaysian Aborigines. Whole blood from twenty-four patients suspected of scrub typhus infection were tested using nested polymerase chain reaction and sera were evaluated by the indirect immunoperoxidase test. Antibody responses towards Rickettsia tsutsugamushi were observed in seventeen patients with the majority having high titers of IgG antibodies. Seven patients were seronegative. The nested PCR amplified R. tsutsugamushi DNA from six patients, of which two were negative serologically and four had high titers of IgG antibodies. Second samples collected seven days after treatment were negative by PCR testing. Nested PCR is highly sensitive and specific and may be used to provide rapid confirmation of scrub typhus cases in endemic region.
A review of the various studies on toxoplasmosis in peninsular Malaysia is presented. The period of review spanned between 1973 and 1980 during which a number of serological surveys were carried out for the presence of Toxoplasma gondii antibody in Malaysians, using either the indirect hemagglutination (I.H.A.) or the indirect fluorescent antibody (I.F.A.) tests. The prevalence rates of Toxoplasma antibody were consistently foundhighest among Malays, followed by Indians, Orang Aslis (Aborigines) and lowest among Malays, followed by Indians, Orang Aslia (Aborigines) and lowest among Chinese, the 4 major ethnic groups living in Malaysia. Positive titres, present in all age groups, showed an increase with age but no difference due to sex. However, higher prevalence of positive cases was recorded among rural dwellers and the lower socioeconomic group than from urban dwellers. The possible routes of infection among the ethnic groups were discussed. Among animal populations, the presence of Toxoplasma antibody was detected in buffaloes, swine, goats, cattle, cats and dogs. The epidemiological importance of the findings are discussed and suggestions made for future studies.
Indirect fluorescent antibody (IFA) tests and enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies to Plasmodium falciparum in an indigenous population in an area of Malaysia with high malaria prevalence. The results of three surveys were analyzed to examine the relation of these serologic measures with age, parasite rate, and spleen size. For children 0-4 years old, increasing spleen size was associated with an increasing likelihood of malaria parasitemia, while for 5-9 year olds the two variables were unrelated. Parasite rate declined with age and ELISA titre increased with age in all surveys; IFA titre was consistently high and did not vary with age. Neither antibody measure was significantly correlated with either the presence or the actual density of parasitemia. These antibody measures are most useful as adjuncts to the more traditional techniques of malaria assessment.
A monoclonal antibody raised against Haemophilus ducreyi was tested for its sensitivity and specificity as an immunofluorescence (IF) reagent using simulated vaginal smears containing H. ducreyi, smears taken from skin lesions of mice infected with H. ducreyi and patients from South Africa, Thailand and Malaysia with clinically diagnosed chancroid. The IF test was more sensitive than culture or Gram staining in the simulated smears, theoretically detecting less than 4 organisms/sample. It detected H. ducreyi in 95% of the animal lesions compared with 14% detected by culture. Immunofluorescence testing identified over 90% of culture-positive cases of chancroid but also detected organisms in some culture-negative cases where clinical evidence for the diagnosis was strong. These results suggest that this antibody may provide a simple, rapid and sensitive means of detecting H. ducreyi in cases of chancroid.
The indirect fluorescence antibody technique has been employed to study the prevalence of toxoplasma antibodies in Singapore. 42.5% of clinically suspected cases of toxoplasmosis showed antibody titres. Of these, 17.5% had titres greater than or equal to 1.64. Malays and Indians have higher positive rates compared to the main ethnic group, the Chinese. Antibody titres are found in both males and females and span through the various age groups. The possible mode of transmission is discussed and the importance of congenital toxoplasmosis is indicated.
A new rat cytomegalovirus (RCMV) isolated from the placenta/uterus of a house rat (Rattus rattus diardii) was found to productively infect rat embryo fibroblast (REF) cells. The virus produced typical herpesvirus-like cytopathic effects characterized by a lytic infection. The well-known herpesvirus morphology was confirmed by electron microscopy. Its slow growth in cell culture indicated that the virus is belonging to subfamily Betaherpesvirinae. Electron microscopy techniques and immunohistochemistry confirmed the presence of herpesviral inclusion bodies and virus related particles in the cytoplasm and nucleus of infected cells. Hyperimmune serum against the Maastricht strain of RCMV revealed the virus identity in neutralization test, immunoperoxidase and immunofluorescence techniques. Despite typical characteristics of CMV, the viral genome is significantly different from that of Maastricht, English, UPM/Sg and UPM/Kn strains. The dissimilarities, which have not been reported before, had been confirmed by mean of restriction endonuclease analysis. The new RCMV strain, a virus that infects placenta and uterus of rats, has been named as ALL-03.
Congenital malaria from Malaysia is reported here for the first time. It occurred in a baby boy born to a 16-year-old primigravida who contracted Plasmodium falciparum infection during pregnancy. She suffered malaria during the later stages of pregnancy and at parturition. The placenta was heavily infested with various asexual stages of P. falciparum. Gametocytes were not seen. Extensive search did not show other species. Cord blood showed very light infection with young trophozoites of P. falciparum. Serological studies using IFA technique showed specific IgG and IgM antibodies to P. falciparum in maternal cord and two early neonatal sera. These serum samples showed lower levels of IgG antibodies against P. vivax and P. malariae, but there were no specific IgM antibodies against these species. The value of specific IgM antibody in the diagnosis of congenital malaria is discussed.