Indirect Immunofluorescence Technique Research Articles

Development of an indirect immunofluorescence technique for the diagnosis of toxoplasmosis in bottlenose dolphins

The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or postmortem features, in humans and other animals. The only diagnostic test described so far for the serological diagnosis of Toxoplasma gondii in marine mammals is the modified agglutination test (Dubey et al., Am J Vet Res 48(8):1239-1243, 1987). The development of more sensible and specific immunological techniques requires specific antibodies, which are currently unavailable in the scientific market. Indirect immunofluorescence (IIF) is one of the most widely used methods for the diagnosis of toxoplasmosis in humans (Auer et al., Parasitol Res 12:965-970, 2000). In order to develop and apply this technique to the bottlenose dolphin (Tursiops truncatus), immunoglobulins were firstly purified using ion-exchange chromatography. The purified immunoglobulins were then injected in New Zealand rabbits in order to obtain polyclonal antibodies. These antisera were validated by the IIF technique, using as controls serum samples of dolphins infected by Toxoplasma. The results were visualized using antirabbit IgG labeled with fluorescein. This newly developed and specific serological assay was then tested with the dolphin collection of Loro Parque, Tenerife, Spain (group I), and L'Oceanogràfic of Valencia, Spain (group II). The obtained results in this study showed that none of the dolphins from group 1 were infected by T. gondii and two animals were positive in group 2. Furthermore, we conclude that this study has produced antibodies with high specificity against dolphin immunoglobulins and an IIF method which may be used as immunological diagnostic tools, especially for the serological diagnosis of toxoplasmosis.

Smoking, disease activity, permanent damage and dsDNA autoantibody production in patients with systemic lupus erythematosus

The aim was to study the association of smoking with the activity and severity of systemic lupus erythematosus (SLE) and the production of antibodies to dsDNA. The study included 223 SLE patients attending the outpatient clinics at Helsinki University Central Hospital. The history of smoking was obtained by personal interview, and clinical data related to SLE by interview, clinical examination and chart review. The activity of SLE was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score and permanent damage by the SLICC/ACR score. Antibodies to dsDNA were determined by three ELISA assays, by the indirect immunofluorescence technique using Crithidia luciliae cells as substrates and by the Farr assay. There were no significant differences in the SLEDAI scores between current smokers (73 patients), ex-smokers (59) and never-smokers (91), though current smokers tended to have lower disease activity. The SLICC/ACR scores between the groups were practically equal. Current smokers had significantly lower levels of antibodies to dsDNA than ex- and never-smokers (p = 0.025). Our study suggests that cigarette smoke may have immunosuppressive effect on autoantibody production in patients with SLE. Permanent damage was not found to be associated with smoking.

3. Cutaneous innervation involvement in CADASIL

Our aim was to study peripheral nerve involvement in CADASIL, a hereditary cerebral microangiopathy due to mutations in the Notch 3 gene. We studied skin innervation in 14 patients with CADASIL (6 women, 8 male, mean age 57.6 ± 5.4) by means of 3 mm-punch biopsy from thigh, distal leg and fingertip. Samples were processed using indirect immunofluorescence techniques and an extensive panel of primary antibodies to mark both myelinated and unmyelinated somatic and autonomic nerve fibers. Vascular and neural signs of remodeling were present in all patients. We found, compared to 40 age and sex matched controls, a lower density of ENF/mm in patient thigh (12.7 ± 4.3 versus 23.4 ± 3.8), leg (6.7 ± 1.3 versus 13.3 ± 3.0) and fingertip (1.8 ± 0.8 versus 6.9 ± 3.1). In fingertip we found also a marked loss of Meissner corpuscles with a lower density/mm2 (8.3 ± 2.2 versus 27.3 ± 7.4). In addition autonomic innervation appeared severely affected with aspect of structural derangement of dermal annexes. Vascular bed appeared very abnormal with dilated and complex vessels. The involvement of last endings of sensory and autonomic nerve fibers is part of the neuropathological picture of CADASIL.

The antigenic character of zinc-binding proteins and their localization in boar reproductive tract--a preliminary study.

In this study immunoelectrophoretic and double immunodiffusion analyses were used to investigate the antigenic character of zinc-binding proteins (ZnBPs), whereas the indirect immunofluorescence technique was used to identify their origin in boar reproductive tract. The mmunoelectrophoretic analysis of ZnBPs of the seminal plasma resulted in the appearance of three antigenic protein complexes, while specific immunoreactivity patterns of the anti-ZnBP serum were detected by double immunodiffusion analysis. Indirect immunofluorescence technique confirmed that ZnBPs were secreted by different reproductive tract tissues, suggesting their contributions to the seminal plasma.

Anti Cathepsin Antibody In Systemic Lupus Erythematosus

Antineutrophil cytoplasmic autoantibodies (ANCA) were found in patients with systemic lupus erythematosus (SLE). Cathepsin G was the major target antigen. However, some ANCA-positive sera did not recognize either of them.The present study was to investigate the unknown target antigens of ANCA (Cathepsin G) in patients with SLE and its relation to the activity of the disease (ds DNA ),C1q ,C3and C4.Seventy four SLE patients referred to immunological department in teaching laboratory\ medical city during period of (1st of march–31st of May) 2011 and 30 apparently healthy individual as a control group were subjected to Antinuclear antibody (ANA), Cathepsin G and C1q were detected by enzyme-linked immunosorbent assay (ELISA) technique.While dsDNA was detected by indirect immunofluorescent (IIF) technique and C3, C4 by single radial immunodiffusion (SRID).The age of SLE patients ranged between (4-71) years with mean age of 32.4. The mean C1q and Cathepsin G levels were 30.14 and 9.7 respectively with significant difference if compare it with the healthy control group. A significant result found between mean age of SLE patients and Cathepsin Ab of a P value 0.007.The Cathepsin G antibody was found in 26 (35%) of SLE patients with a significant P value (0.0001).The Cathepsin Ab were detected in 18 (43.9%) of a patients with positive ds DNA with no significant P value 0.078. while C1q Ab were detected in 38 (51.4%) of SLE patientsand only 18 (47.4%) from them with Cathepsin Ab with statistically significant P value 0.024.Cathepsin Ab found in 19 (42.2%) of SLE patients with low level of C3 and only 6 (21.4%) of normal level of C3 with no significant P value0.076.This study showed 54(73.0%) of SLE patients had low level of C4 with no significant P value (0.15) and the Cathepsin Ab detected in 22 (40.7%) of them with no statistically significant P value 0.097.A statistically significant result were found for ANA, C1q,C3,C4 and Cathepsin Ab if we compare the result of SLE patients with control group, while The Cathepsin G antibodywas found statistically significant in SLE patients ,with significant relation to C1q,on other hand no relation found with the activity of the disease (ds DNA ),C3 and C4 levels.

Modulation of serum smooth muscle antibody levels by levamisole treatment in patients with oral lichen planus

Serum autoantibodies have been found in patients with oral lichen planus (OLP). This study evaluated whether OLP patients had significantly higher frequencies of serum smooth muscle antibody (SMA) than healthy control individuals, and assessed whether levamisole treatment could modulate the serum SMA levels in OLP patients. This study used an indirect immunofluorescence technique to measure the baseline serum SMA levels in a group of 647 OLP patients and 53 controls. Ninety-five SMA-positive OLP patients were treated with levamisole under a regular follow-up schedule, and their serum SMA levels were measured after treatment. The frequencies of serum SMA in patients with OLP (21.9%), erosive OLP (EOLP, 21.6%), major EOLP (17.9%), minor EOLP (24.2%), and nonerosive OLP (24.4%) were all significantly higher than that (0%) in healthy controls (all p<0.001). Of 142 SMA-positive OLP patients, 95 were treated with levamisole under a regular follow-up schedule. Treatment with levamisole for a period of 2-29 months (mean, 9.4±6.0 months) effectively reduced the high mean serum SMA titer (71.0±7.2) at baseline to an undetectable level (0) in all SMA-positive OLP patients, regardless of different initial serum SMA titers. There was a significantly higher frequency of serum SMA (21.9%) in OLP patients than in healthy controls. Treatment with levamisole for 2-29 months significantly reduced the high serum SMA to an undetectable level, and significantly improved the signs and symptoms in all treated OLP patients.

Presence and distribution of FMRFamide-like immunoreactivity in the sea cucumber Holothuria scabra (Jaeger, 1833)

Since the discovery of FMRFamide, a cardioactive neuropeptide in the mollusk Macrocallista nimbosa (Lightfoot, 1786) by Price and Greenberg (Science 197:670–671, 1977), there has been a steady increase in our understanding of the distribution and diversity of FMRFamide and FMRFamide-related peptides (FaRPs) across phyla. The sea cucumber Holothuria scabra (Jaeger, 1833) (Echinodermata: Holothuroidea) is a high-premium tropical economic species that is overexploited globally and may be in imminent danger of extinction in some areas. Conservation of this species and its amenability to culture is however hampered by the limited knowledge about its biology, including the neurohormonal system. Using indirect immunofluorescence technique with antisera raised against the tetrapeptide FMRFamide as well as reverse-phase HPLC, we have demonstrated the widespread distribution of FMRFamide-like immunoreactivity in the tissues of H. scabra, including the coelomic fluid, suggesting that FaRPs might have neurohormonal activities in this species.

Prevalence and risk factors for Toxoplasma gondii infection among pregnant and postpartum women attended at public healthcare facilities in the City of Niterói, State of Rio de Janeiro, Brazil

To determine the prevalence of immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-Toxoplasma gondii antibodies among pregnant and postpartum women attended within the public healthcare system in Niterói, State of Rio de Janeiro,and to detect possible exposure factors associated with T. gondii infection in this population. IgM and IgG anti- T. gondii antibodies were investigated in 276 pregnant and 124 postpartum women by using the indirect immunofluorescence (IFAT) and immunoenzymatic assay (ELISA) techniques. The participants were selected by convenience sampling. All these 400 patients filled out a free and informed consent statement, answered an epidemiological questionnaire and were informed about the disease. Among the 400 samples analyzed, 234 (58.5%) were reactive to IgG anti-T. gondii antibodies, according to the IFAT and/or ELISA assay. One pregnant woman was found to be reactive to IgM anti- T. gondii antibodies, with an intermediate IgG avidity test. Risk factor analysis showed that seropositivity was significantly associated (p<0.05) with age, contact with cats and presence of rodents at home. Through a logistic regression model, these associations were confirmed for age and contact with cats, while education at least of the high school level was found to be a protective factor. The prevalence rate of IgG anti-T. gondii antibodies in the city of Niterói was high and the risk factors for infection detected after multivariate analysis were: age over 30 years, contact with cats and education levels lower than university graduate level.

Does anti-DNA positivity increase the incidence of secondary antiphospholipid syndrome in lupus patients?

Aim of the workTo detect the incidence of secondary antiphospholipid syndrome (APS) among Systemic lupus erythematosus (SLE) patients with positive anti-DNA antibodies. Patients and methodsWe studied 342 SLE patients; Group I: anti-DNA positive SLE patients (n=208) and Group II: anti-DNA negative SLE patients (n=134), with a female to male ratio of 9.39:1 and a mean age of 27.49±7.94years and disease duration of 5.74±3.97years. Full history taking, thorough clinical examination, laboratory and relevant radiological investigations were performed. Disease activity was assessed using systemic lupus erythematosus disease activity index (SLEDAI). Anti-dsDNA tests were carried out by indirect Immunofluorescence (IF) technique. Anti cardiolipin antibodies (IgG and IgM) and Anti-β2 glycoprotein-I antibody of IgG and/or IgM isotype were detected by ELISA. ResultsThe clinical manifestations, disease activity and laboratory investigations of the SLE patients varied according to the anti-DNA antibodies. Thirty-six patients (17.3%) had secondary APS in those with positive anti-DNA antibodies while only16 (11.9%) had secondary APS in those with negative anti-DNA antibodies, with no significant differences between both groups. ConclusionApparent higher incidence of secondary APS was detected in anti-DNA positive SLE patients. The non significant differences between both groups may suggest that anti-DNA positivity cannot be considered as the only predictor of secondary APS and further studies may be needed to detect other factors which may increase the incidence of APS in SLE patients.

Evaluation of seroepidemiological toxoplasmosis in HIV/AIDS patients in the south of Brazil

Toxoplasmosis is considered one of the opportunistic infections for individuals with the Acquired Immunodeficiency Syndrome (AIDS), and is also a major cause of morbidity and mortality. The aim of this study was to evaluate the prevalence of neurotoxoplasmosis, ocular toxoplasmosis and antibodies for Toxoplasma gondii in HIV-positive patients attending the SAE (Specialized Assistance Service for HIV/AIDS), as well as to associate their serological profile with epidemiological and clinical data. A total of 250 patients participated in the study from December, 2009 to November, 2010. Serological analysis was performed using the indirect immunofluorescent technique; epidemiological data were gathered by a questionnaire, and clinical history was based on the analysis of medical charts. Prevalence of seropositivity was 80%, with history of neurotoxoplasmosis in 4.8% and of ocular toxoplasmosis in 1.6% of the patients. The Highly Active Antiretroviral Treatment (HAART) was not used by 32% of the patients, 18.4% of the patients had CD4+ T- lymphocyte count less than 200 cells/mm³ and 96.8% of them were not aware of the modes of disease transmission. These findings led us to conclude that the study population is at high risk of clinical toxoplasmosis, because of both reactivation of infection in the seropositive patients who do not make a regular use of HAART, and primo-infection in seronegative patients worsened by an unawareness of the modes of infection reported in this study.

Systemic sclerosis: demographic, clinical and serological features in 100 Iranian patients

To evaluate demographic, clinical and laboratory features associated with scleroderma-specific auto-antibodies. Sera of 100 patients with systemic sclerosis (SSc) were analyzed by an indirect immunofluorescence technique with HEp-2 cells as a substrate. Specific ANA such as anti-centromere antibodies (ACA), anti-topoisomerase (TOPO), anti-RNA polymerase III (Pol 3), anti-U3-RNP (U3-RNP), anti-Th/To (Th/To) and anti-PM/Scl (PM/Scl) were detected by line immunoassay and anti-U1-RNP (U1-RNP) by ELISA. Frequency of clinical features associated with a specific antibody group was reported cumulatively over the follow-up period. Frequency of specific clinical features was compared across the two disease subtype including limited cutaneous (lcSSc) or diffuse cutaneous (dcSSc) as well as the auto-antibody groups. Ninety-four percent of patients were ANA positive with significant higher skin score, Raynauds and digital ulcer/gangrene. Anti-TOPO was detected in 71% of all patients, in 90.5% of dcSSC and in 65.8% of lcSSc. Anti-TOPO was significantly associated with dcSSc, higher skin score, digital ulcer/gangrene, pulmonary fibrosis, DLCO <70%. U1-RNP antibody was associated with lower fibrosis in lung. ACA was positive in 7% of patients and exclusively in those with lcSSc. We did not find association between gender and presence of auto-antibodies. Anti-TOPO antibody had a high prevalence in contrast to low prevalence of ACA antibody. There were no differences in clinical subtypes of the disease in patients with positive anti-TOPO and positive ACA. Differences in prevalence of auto-antibodies are suggestive of further genetic study.

THE ROLE OF THE PEPTIDE INTERMEDIN IN A NOVEL MODEL OF PULMONARY HYPERTENSION

IntroductionPulmonary hypertension (PH) is characterised by elevated pulmonary arterial pressures, inappropriate apoptosis and vascular remodelling. Animal models fail to replicate such cellular changes. Current therapies target several mediators including endothelin-1...

Phenotype, differentiation, and function differ in rat and mouse neocortical astrocytes cultured under the same conditions

The study of slowly progressing brain diseases in which glial cells play a pathogenic role requires astrocytes that have been cultured for several weeks. We characterized neocortical astrocytes, grown for up to 42 days in vitro (DIV), from newborn rats and mice by indirect immunofluorescence technique, Western blot, and real-time RT-PCR analyses. We obtained highly enriched rat and mouse astrocyte cultures, where most cells were positively stained for the astrocyte markers GFAP, vimentin, and S100β, whereas neuronal and oligodendrocyte markers were undetectable. The protein and mRNA levels of GFAP, vimentin, and nestin were higher in rat than in mouse astrocytes. From 28 to 42 DIV, the levels of vimentin and nestin, but not of GFAP, decreased in both species, with an increase in the vimentin-GFAP ratio of 1.7 for rat, and of 0.9 for mouse astrocytes suggesting that the rat cultures were more differentiated than the mouse cultures, although both remained partially immature. The protoplasmic appearance of the cells, the negative A2B5 immunoreactivity, and the expression of the glutamate transporters GLAST and GLT-1 indicate that the rat and mouse cultures contained mainly type I astrocytes. The protein levels of GLAST and GLT-1 decreased from 28 to 42 DIV in the mouse, but not in the rat astrocytes, suggesting that the rat cultures are suitable for functional studies. Thus, under the same culture conditions, astrocyte cultures from rats and mice differ in phenotype, differentiation, and functionality. This finding should be taken into account when long-lasting glial reaction patterns are being studied.

Human TTRV30M localization within podocytes in a transgenic mouse model of transthyretin related amyloidosis: does the environment play a role?

Transthyretin related amyloidosis is a nosological entity that leads to disability, diminished quality of life, all stages of chronic kidney disease and eventually death. Podocytes are polarized, highly differentiated epithelial cells important for proper nephron function. In the present study we investigated whether deposited TTRVal30Met (TTRV30M) molecules could be localized within podocytes in situ under the effect of different housing conditions (i.e. specific pathogen free [SPF] vs. non-SPF). Murine renal glomeruli from human TTRV30M (hTTRV30M) transgenic mice were examined via direct and indirect immunofluorescence techniques for the presence of hTTRV30M, murine serum amyloid P, activated caspase-3 and NPHS1. Association strength and amount of colocalization for NPHS1-hTTRV30M, NPHS1-activated caspase-3, hTTRV30M-murine serum amyloid P were estimated. Localization of hTTRV30M in podocytes was demonstrated by immuno-electron microscopy. Renal hTTRV30M gene and NPHS1 gene expression levels were estimated. Non-SPF transgenic mice showed increased glomerular hTTRV30M deposition compared to their SPF counterparts. Furthermore increased podocytic localization of hTTRV30M was noticed in non-SPF mice. Glomerular caspase-3 activation was increased only in the non SPF housing conditions. Podocytic caspase-3 activation was increased in SPF and in non-SPF transgenic mice when compared to non transgenic controls. Environmental conditions influence glomerular deposition and podocytic localization of hTTRV30M. In this context increased caspase-3 activation occurred.

Automation in indirect immunofluorescence testing: a new step in the evolution of the autoimmunology laboratory.

Indirect immunofluorescence (IIF) plays an important role in immunological and immunometric assays for detecting and measuring autoantibodies. This technology was the first multiplex method used to detect cardinal autoantibodies for the diagnosis of autoimmune diseases. Over the last 20 years, research has enabled the progressive identification of cell and tissue autoantigens which are the target of autoantibodies originally detected by IIF. Accordingly, newer immunometric methods, capable of measuring concentrations of specific autoantibodies directed against these autoantigens, allowed for a gradual replacement of the IIF method in the autoimmunology laboratory. Currently, IIF remains the method of choice only in selected fields of autoimmune diagnostics. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of ANA, the biomedical industry has developed technological solutions which significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. This review summarizes the general and specific features of new available commercial systems (Aklides, Medipan; Nova View, Inova; Zenit G Sight, A. Menarini Diagnostics; Europattern, Euroimmun; Helios, Aesku.Diagnostics; Image Navigator, Immuno Concepts; Cytospot, Autoimmun Diagnostika) for automation of the IIF method. The expected advantages of automated IIF are the reduction in frequency of false negative and false positive results, the reduction of intra- and inter-laboratory variability, the improvement of correlation of staining patterns with corresponding autoantibody reactivities, and higher throughput in the laboratory workflow.

High HBcAg Expression in Hepatocytes of Chronic Hepatitis B Patients in Bangladesh

The present study evaluates the intracellular expression of the hepatitis B Virus (HBV) core antigen in chronic hepatitis B (CHB) patients from Bangladesh in relation with serological, virological and histological variables. A cross-sectional study was carried out among 70 patients incidentally diagnosed as HBV chronic carriers who have undergone liver biopsy. Indirect immunofluorescence technique was used to study the intracellular expression of hepatitis B core antigen (HBcAg). The grade and cellular distribution were semiquantitatively scored according to the percentage of stained cells and their nuclear, cytoplasmic or mixed expression pattern respectively. A higher HBcAg grade was related to higher viral load, alanine aminotransferase levels and histological damage. Higher HBV DNA counts and HBcAg grade were found in hepatitis B e antigen (HBeAg)-positive patients. Abbreviations: HBV: Hepatitis B virus; CHB: Chronic hepatitis B; HBcAg: Hepatitis B core antigen; HBeAg; Hepatitis B e antigen.

Down-regulation of hematopoiesis master regulator PU.1 via aberrant methylation in chronic myeloid leukemia

The PU.1 transcription factor is a crucial regulator of hematopoiesis, and its expression is altered in various leukemic processes. It has been shown that expression of PU.1 is severely impaired in patients with chronic myeloid leukemia (CML), but the mechanism underlying this effect remains unknown. Through bisulfite sequencing, semi-quantitative PCR, and indirect immunofluorescence and Western blot techniques, we found aberrant methylation in the promoter region of transcription factor PU.1 in CML patients both in the chronic and blast crisis phases, as well as in the CML blast K562 cell line. Of these, several CpG sites were more highly methylated in blast crisis than chronic phase, while no methylation of these sites was observed in healthy individuals. Interestingly, CML patients achieved complete cytogenetic remission under imatinib mesylate treatment, but the aberrant methylation status of PU.1 was not reversed. Down-regulation of PU.1 expression at the mRNA and protein levels was also observed in association with aberrant methylation. Thus, for the first time, we have revealed a potential epigenetic modification of PU.1 in CML, which may be responsible for the down-regulation of PU.1. These data suggest that aberrant methylation of PU.1 may play a role in CML pathogenesis, and may therefore serve as a useful biomarker and potential target for demethylating drugs.

Antineutrophil Cytoplasmic Autoantibodies: How Are They Detected and What Is Their Use for Diagnosis, Classification and Follow-up?

Antineutrophil cytoplasmic antibodies (ANCA) are traditionally detected by an indirect immunofluorescence technique. According to the international consensus on ANCA testing, ANCA should also be tested by antigen-specific tests for myeloperoxidase-ANCA and proteinase 3-ANCA. The direct noncompetitive enzyme-linked immunosorbent assay (ELISA) used to be the method of choice. Nowadays, these assays are called "first-generation" assays. Second-generation tests (capture ELISA) or third-generation tests (anchor ELISA) are more sensitive and specific for ANCA testing. We postulate that ANCA as detected by these newer ANCA tests may replace the need to perform indirect immunofluorescence-based assays. For classification of patients, ANCA serotype seems more important than classifying patients according to their clinical subtype, since genetics, clinical manifestations and response to therapy are more related to ANCA serotype than to clinical subtype. Detection of ANCA to monitor disease activity is still a controversial issue. Treatment based on ANCA levels is at present only experimentally performed in those patients who are treated with B-cell depletion therapy with rituximab. Future studies are needed to establish whether this way of monitoring patients is warranted.

Expressions of vascular endothelial growth factor and its receptors in rat conjunctival fibroblasts

To detect the expressions of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFR) in cultured rat conjunctival fibroblasts. Experimental study. Conjunctiva was obtained from each eye of five Sprague Dawley (SD) rats under local anesthesia. Tissues were minced and grown in culture flask containing Dulbecco's Modified Eagle's medium (DMEM). The fibroblasts were identified by observing cell morphology, and the expression of staining of cytokeratin and vimentin by inverted microscopy and indirect immunofluorescence technique. mRNA primers were designed for VEGF(164), VEGFR-1 (Flt-1), and VEGFR-2 (Flk-1). mRNA was extracted by Trizol method. cDNA was synthesized under the action of RNA reverse transcriptase. Ladder-like pattern of DNA fragmentation appeared upon 2% agarose gel electrophoresis. The amplification curves and dissociation curves of VEGF(164), VEGFR-1(Flt-1), and VEGFR-2 (Flk-1) were detected by realtime polymerase chain reaction (PCR). The expression of VEGFR-1 on fibroblast was observed by immunofluorescence technique. The fluorescent staining was negative in cytokeratin of fibroblasts, but was positive in vimentin of fibroblasts. The mRNA expressions of VEGF(164), VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) were detected in fibroblast and verified by gene sequencing. The peaks of the dissociation curves were all steep and specific. The fluorescent staining of VEGFR-1 was positive. Our results demonstrate that the expression of VEGF, VEGF receptors, and VEGFR-1 cytokine in rat conjunctival fibroblasts suggests that anti-VEGF compounds may exert a direct influence to the growth of rat conjunctival fibroblast.

Evaluation of in vitro antiviral activity of Chelidonium majus L. against herpes simplex virus type-1

Chelidonium majus Linnaeus (L) has been used in a variety of practical applications in medical sciences. Its antiviral activity against herpes simplex virus type-1 (HSV-1) was investigated in cell culture. C. majus L. extract was found to be non-toxic to Vero cells (African green monkey kidney cells) up to concentration 1000 mg/ml and inhibited the growth and development of HSV-1 in dose-dependent manner in Vero cells. In order to study the possible mechanisms of the antiviral activity of C. majus L. extract, cells were treated with extract before, during and after infection, and the viral titers were tested by TCID50 assay. The antiviral effects in treatment of post and during virus infections were more remarkable than the treatment of pre-infection. For further investigation indirect immunofluorescence technique was used to elucidate the antiviral mechanism of the extract by infecting Vero cells at different times and monitoring the synthesis of viral proteins. Although the precise mechanism has not yet to be defined, our work indicated that C. majus L. extract could inhibit growth and development of HSV-1 in cells in vitro. Key words: Chelidonium majus L, herpes simplex virus type-1 (HSV-1), antiviral activity, Vero cell, TCID50.