Duration of syphilis symptoms at presentations in men who have sex with men in Australia: are current public health campaigns effective?

The rapid rise in syphilis cases has prompted a number of public health campaigns to assist men who have sex with men (MSM) recognize and present early with symptoms. This study aimed to investigate the temporal trend of the duration of self-report symptoms and titre of rapid plasma reagin (RPR) in MSM with infectious syphilis. Seven hundred and sixty-one syphilis cases in MSM diagnosed at the Melbourne Sexual Health Centre (MSHC) from 2007-2013 were reviewed. Median duration of symptoms and RPR titres in each year were calculated. The median durations of symptoms with primary and secondary syphilis were 9 [interquartile range (IQR) 6-14] days and 14 (IQR 7-30) days, respectively. The overall median titre of RPR in secondary syphilis (median 128, IQR 64-256) was higher than in primary syphilis (median 4, IQR 1-32) and in early latent syphilis (median 32, IQR 4-64). The median duration of symptoms for primary syphilis, secondary syphilis and titre of RPR level did not change over time. Public health campaigns were not associated with a significant shorter time from onset of symptoms to treatment. Alternative strategies such as more frequent testing of MSM should be promoted to control the syphilis epidemic in Australia.

Reliability and factor analysis of children's fear survey schedule-dental subscale in Indian subjects.

CONTEXT: Fear to visit a dentist is a common observation even in adults; however, among children it becomes one of the most important issues for a dentist. Psychographic analysis of the factors that add to fear level of the children can be accessed through Children fear survey schedule-dental subscale (CFSS-DS); however, its varied applicability in different environmental situations has been tested through this paper. AIMS: The aim of present study is to evaluate the reliability and factor structure of the Indian version of the CFSS-DS. MATERIALS AND METHODS: The routine patients attending Outpatient Department of Pedodontics with Preventive Dentistry, Faculty of Dental Sciences, Lucknow, India (n=197, aged 7-12 years old) were evaluated for children's fear survey schedule-dental subscale which was filled by parents on behalf of the child. STATISTICAL ANALYSIS: Reliability analysis (alpha) was performed to assess the internal consistency of the Indian translation of the scale. Factor analysis (principle components, varimax rotation) was employed to assess the factor structure. RESULTS: Children fear survey scale-dental subscale was found to be equally reliable (Cronbach alpha = 0.92) and applicable among Indian subjects. However, factorization revealed emergence of 1) hospital, injections and hospital personnel, 2) drilling and interaction with unknown, 3) dental care personnel and practices. CONCLUSION: The present study extended the universal applicability of children fear survey schedule -dental subscale, while at the same time it was able to highlight different facets of problem in different environments.

Glutamine synthetase localization in cortisol-induced chick embryo retinas.

We report here for the first time, in chick retina, Muller cell localization of glutamine synthetase (GS) activity by an immunohistochemical technique, in agreement with previous reports of glial localization of this enzyme in rat brain and retina. Age-dependent changes in the endogenous enzyme activity as well as cortisol-induced changes in GS activity, both in ovo and in vitro, measured biochemically, reflect the changes observed by staining.

Role of casein on induction and enhancement of production of a bacterial milk clotting protease from an indigenously isolated Bacillus subtilis.

AIMS: To isolate and enhance the yield of a bacterial milk clotting protease (MCP) through process optimization and scale up. MATERIALS AND RESULTS: Bacillus subtilis was isolated as MCP producer with good milk clotting activity (MCA) per proteolytic activity (PA) index. The enzyme production was inducible with casein and enhanced with fructose and ammonium nitrate resulting in 571.43 U ml(-1) of enzyme. CONCLUSIONS: Medium containing 4% fructose, 0.75% casein, 0.3% NH(4)NO(3) and 10 mmol l(-1) CaCl(2), pH 6.0, inoculated with 4% (v/v) inoculum, incubated at 37 degrees C, 200 rev min(-1) for 72 h gave maximum production. A 6.67-fold increase in MCP yield with very high MCA per PA index was observed after final optimization indicating similarity to rennets. SIGNIFICANCE AND IMPACT OF THE STUDY: Mostly fungal MCPs have been reported. The MCA and MCA per PA index of this bacterium is comparable to that of many fungal reports and better than quite a few bacterial MCPs. Thus, this enzyme by B. subtilis has good probability of successful use in cheese production.

Comparative effects of adriamycin and N-trifluoroacetyladriamycin-14-valerate on cell kinetics, chromosomal damage, and macromolecular synthesis in vitro.

N-Trifluoroacetyladriamycin-14-valerate differs from Adriamycin in its rapid intracellular transport and lack of fluorescent binding to nuclei or chromosomes. Both of these anthracyclines cause inhibition in the incorporation of labeled precursors into nucleic acids, extensive chromosomal damage, and arrest of cell cycle traverse in G2. In human lymphoid cells, N-trifluoroacetyladriamycin-14-valerate, unlike Adriamycin, does not show cell cycle phase-specific or proliferation-related cytotoxic effects. In an L1210 soft-agar assay, both Adriamycin and N-trifluoroacetyladriamycin-14-valerate show no enhanced sensitivity of mid-S-phase cells to their cytotoxic action.

Establishment of human retinal pigment epithelial cell lines by oncogenes.

The primary human retinal pigment epithelial cells were transfected with oncogenic sequences derived from viruses and cellular homologues of retroviral oncogenes 'protooncogenes' linked to simian virus 40 (SV-40) and retroviral promoters. Foci of cells were noted between 2 to 4 weeks after transfection. Individual colonies of cells were expanded from cultures transfected with SV-40 virion DNA, SV-40 large T antigen gene, Ha-ras oncogene, human and mouse c-myc and adenovirus E1A gene. Established cell lines tested were positive for the specific oncogene sequences by Southern hybridization and also expressed the protein as assayed by immunofluorescence and immunoblot analysis. Cell lines established with SV-40 large T antigen, and SV-40 virion DNA, exhibited epithelioid morphology up to the 25th passage and later became more rounded. However, all cell lines established with other oncogenes continued to retain epithelial morphology. Functional analysis of the cell lines demonstrated the presence of polarity and the ability to phagocytize rod outer segments, characteristics of retinal pigment epithelial cells. The use of oncogenes with immortalization/transformation potential may allow the establishment of cell lines from ocular tissues for analysing the biochemical basis of a disease like retinitis pigmentosa.

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

Establishment and characterization of a retinal Müller cell line.

PURPOSE: Primary cultures of Müller cells have proven useful in cell biologic, developmental, and electrophysiological studies of Müller cells. However, the limited lifetime of the primary cultures and contamination from non-neural cells have restricted the utility of these cultures. The aim of this study was to obtain an immortalized cell line that exhibits characteristics of Müller cells. METHODS: Primary Müller cell cultures were prepared from retinas of rats exposed to 2 weeks of constant light. Cells were immortalized by transfection with simian virus 40. Single clones were obtained by repeatedly passaging cells using cloning wells. Immunocytochemical and immunoblotting studies were carried out with glial fibrillary acidic protein (GFAP)-specific and cellular retinaldehyde-binding protein (CRALBP)-specific antibodies. Transient transfections with CRALBP-luciferase constructs were performed by electroporation. RESULTS: Oncogene transformation resulted in the establishment of a permanent cell line that could be readily propagated. Immunocytochemical and immunoblotting studies demonstrated that the Müller cell line, rMC-1, expressed both GFAP, a marker for reactive gliosis in Müller cells, and CRALBP, a marker for Müller cells in the adult retina. Transient transfection assays showed that promoter-proximal sequences of the CRALBP gene were able to stimulate reporter gene expression in rMC-1. CONCLUSIONS: Viral oncogene transformation has been successfully used to isolate a permanent cell line that expresses Müller cell phenotype. The rMC-1 cells continue to express both induced and basal markers found in primary Müller cell cultures as well as in the retina. The availability of rMC-1 should facilitate gene expression studies in Müller cells and improve our understanding of Müller cell-neuron interactions.

Cell fate decisions in a human retinal precursor cell line: basic fibroblast growth factor- and transforming growth factor-alpha-mediated differentiation.

The purpose of this study was to determine if immortalized human retinal precursor cells could serve as a model to investigate cues that modulate cell fate and differentiation. We investigated the effects of a variety of growth factors broadly but specifically tested the effects of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)a in retinal cell differentiation and commitment. To determine the role of exogenously added growth factors in a human retinal precursor cell line (KGLDMSM), established from a first-trimester retina, cells were adapted to grow in a defined medium and exposed to a variety of trophic factors (epidermal growth factor [EGF], neuron growth factor [NGF], TGFalpha, TGFbeta, acidic FGF, and bFGF). Dose-response curves were developed to arrive at optimal concentrations. The neurotrophic potential of growth factors was determined by 3H-thymidine incorporation and bromodeoxyuridine (BrdU) labeling. The identity of the emerging neuronal phenotypes were determined by phase-contrast microscopy, immunolabeling for the neuron-specific antigens neurofilament protein (NF) and neuron-specific enolases (NSE), and photoreceptor-specific antigens (Rho1D4, 7G6) using immunocytochemistry and Western blot analysis. To identify some of the early response genes (c-fos, c-myc) expressed in response to growth factors, Northern blot analysis was performed. Almost all of the factors tested increased the total number of cells with a neuronal phenotype. Potency of growth factors to generate neurons was TGFalpha > bFGF > EGF > NGF. Both TGFalpha and bFGF, alone or in combination, increased the total number of neurons. Most of the neurons generated were photoreceptors, as depicted by the polarized phenotype, expression of photoreceptor-specific antigens, and processes resembling rudimentary outer segments. The increase in photoreceptor-like neurons is possibly attributable to an increase in numbers rather than greater survival. Additionally, the majority of the photoreceptors generated labeled with BrdU and for photoreceptor-specific antigens, suggesting that an inductive effect of bFGF and TGFalpha could occur in the cell cycle or shortly thereafter. Both bFGF and TGFalpha induced the expression of the early response gene c-fos while not altering the expression of c-actin or c-myc. The emergence of a photoreceptor phenotype was confirmed by both immunocytochemistry and Western blot analysis. The immortalized retinal precursor cell line could prove valuable in determining the role of exogenously added growth factors in retinal development and differentiation. Both bFGF and TGFalpha enhance the photoreceptor phenotype in medium-density cultures under conditions of defined medium. The same was confirmed by phase-contrast microscopy, immunocytochemistry, and Western blot analysis. Furthermore, cell fate determination in cultured precursor cells could occur during the late part of the cell cycle or shortly after completion of cell division. The effects of TGFalpha and bFGF seem to be slightly additive. The cell line will be extremely valuable in studying mechanisms of cell commitment and generation of retinal cell types, which could be tested for their potential for transplantation.

Transdifferentiation of adult human pigment epithelium into retinal cells by transfection with an activated H-ras proto-oncogene.

The identification of homologs to viral oncogenes in normal cells coupled with development of techniques for DNA transfer into cells offers a powerful approach to dissect the processes associated with differentiation-specific oncogenes. We have derived cell lines by transfection of viral DNAs and proto-oncogenes into primary retinal pigment epithelial (RPE) cells. Establishment of cell lines was successfully achieved with the SV40 large T-antigen gene activated form of Harvey (H)-ras proto-oncogene, c-myc, and adenovirus E1A. The cell lines derived using the H-ras oncogene appeared to contain cells with a neuronal phenotype. This feature was not observed in cell lines established with the other oncogenes. Characteristically, H-ras-transfected cells all exhibited features associated with neurons around 10-14 passages. The transdifferentiated cells were biochemically characterized and found to express neuronal markers, such as neurofilament protein and neuron-specific enolases. The specific neuronal changes were restricted to only two primary cultures of RPE derived from carcinoma donors. Although transdifferentiation of pigmented cells of iris, or the retina, into the lens has been demonstrated, our studies presented in this report provide evidence that RPE cells from adults can transdifferentiate into neurons under the influence of a specific oncogene. To the best of our knowledge, this is the first report on transdifferentiation of adult human pigment epithelium into a neuronal cell type.

Establishment of a human retinal cell line by transfection of SV40 T antigen gene with potential to undergo neuronal differentiation.

Recently, a number of laboratories have been interested in developing cell lines of ocular tissues to understand the pathogenesis of ocular diseases. Toward this end, we report here the generation of cell lines of human retina by transfection of simian virus SV40 T antigen gene. Established retinal cells grow as a monolayer and exhibit limited serum dependence. Phase-contrast and electron microscopic studies revealed distinct morphological cell types. Immunofluorescence studies showed that the established retinal cells were positive for neuron-specific enolase, neurofilament protein, glycine receptor, synaptophysin, and secretogranin. Cells were negative for glial fibrillary acidic protein, glutamine synthetase, galactocerebroside, and carbonic anhydrase II. In addition to neuronal features, a small percentage of flat cells were, however, positive for cellular retinaldehyde binding protein, and cells with the phenotype of rod and cone photoreceptor coexpressed opsin and interphotoreceptor retinoid-binding protein. An important feature of this cell line is that addition of phorbol ester and cAMP induced dramatic changes, with 100% of the cells extending long, thin neuritic processes. Thus, the established retinal cells would be useful for studies dealing with differentiation and plasticity of the cells of the nervous system.

The catecholamine content of a cystic phaeochromocytoma.

We report a case of phaeochromocytoma in which the tumour was a single 10 cm adrenal cyst. The cyst fluid contained noradrenaline and adrenaline in concentrations 100 to 200 fold higher than are seen in the serum of phaeochromocytoma patients. Possibly cystic tumours containing large amounts of catecholamine may if inadvertently compressed at operation, present a greater surgical hazard than solid tumours.

Nucleoside transport sites in a cultured human retinal cell line established by SV-40 T antigen gene.

Adenosine, an important neuromodulatory compound in the brain and retina, is a potent vasodilator in most vascular beds throughout the body. Its actions are potentiated by inhibitors of nucleoside transport into cells. Knowledge of the existence of specific adenosine uptake systems in mammalian retina and the inhibition of the uptake by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, raises the possibility that the associated nucleoside transport system may be of pharmacological importance in retinal function. We have characterized the binding of the nucleoside transporter probe, [3H]NBMPR, to a cultured human retinal cell line established by transfection of SV-40 T antigen plasmid-DNA. The binding was specific, saturable and reversible. Scatchard analysis of the saturation data revealed that NBMPR binds to a homogeneous population of high affinity binding sites (KD = 0.65 +/- 0.22 nM; Bmax = 466 +/- 157 fmol/mg protein) characteristically similar to the binding sites in human retinal tissue (KD = 0.32 +/- 0.01 nM; Bmax = 292 +/- 41 fmol/mg protein). Selected compounds inhibited the binding in the cell line and retinal tissue with the same rank order of potency, suggesting that the transporters in the cell line and retinal tissue are similar. The data showed that the cell line is a useful model for the study of nucleoside transporter function in human retina.

Proto-oncogene expression in cAMP and TPA-mediated neuronal differentiation in a human retinal cell line KGLDMSM.

PURPOSE: A human retinal cell line, KGLDMSM, developed by SV-40T antigen gene transfection, is stable in culture for a long period, unlike the primary cells. The cell line shows some degree of morphological differentiation with limited extension of stublike neurites upon transfer to defined medium. In our effort to explore genes implicated in neuritic extension and neuronal differentiation seen in response to cAMP and TPA, we have analyzed time dependent induction of a variety of proto-oncogenes: c-myc, H-ras, c-ras, and c-fos. METHODS: Cells were adapted to grow in defined media and exposed to differentiation inducing agents cAMP, TPA, Retinoic Acid, and sodium butyrata. Cells were assessed for phenotypic changes and altered expression of proto-oncogenes as evaluated by Northern Blot analysis and immunocytochemistry. RESULTS: Exposure of the cells to cAMP and TPA induced dramatic changes, with 100% of the cells extending neuritic processes. However, other differentiation inducing agents such as retinoic acid and sodium butyrata failed to elicit any response. We report that agents that promote neuritic extension also induce expression of c-fos. Transcriptional activation of c-fos in response to cAMP (30 min) and TPA (1hr) is also accompanied by expression of fos gene product as evaluated by using fos antibody. No fos expression was seen in uninduced cells. CONCLUSION: In retinal cell line KGLDMSM, agents that enhance neuronal differentiation (cAMP, TPA) also induce c-fos expression. Expression of c-fos may be a necessary prerequisite in neuronal differentiation and the established retinal cell line offers an excellent cell model for dissecting the molecular events underlying neuronal differentiation.

In vitro phenotypic and functional characterization of human pigment epithelial cell lines.

We have characterized human retinal pigment epithelium (HRPE) for the expression of cell surface antigens. Primary HRPE cultures, established cell lines, and freshly brushed pigment epithelial cells all express HLA-ABC but not HLA-DR antigens. However, both primary cultures and established cell lines can be induced by gamma interferon stimulation to express HLA-DR in a dose dependent manner. Only freshly brushed HRPE cells express Fc, and no cells demonstrated the presence of C3b. Our results show that HRPE cells change in culture, as reflected by the loss of Fc receptors, but retain the ability to synthesize HLA-ABC spontaneously and HLA-DR upon stimulation.

Extracellular matrix mediated growth and differentiation in human pigment epithelial cell line 0041.

Efforts to grow differentiated pigment epithelial cells have led to a characterization of the growth kinetics of spontaneously established, continuously growing, human retinal pigment epithelial (PE) cell line 0041 on several biomatrices. These substrates were prepared from (a) placental and amniotic membrane, (b) commercially available basement membrane matrix (Matrigel), (c) dishes coated with extracellular matrix secreted by endothelial cells (ECM), (d) dishes coated with collagen IV and/or laminin, (e) dishes coated with collagen I and/or fibronectin. Our findings suggest that tissue culture plastic and dishes coated with collagen IV alone promote higher cell densities, while highest plating efficiency (24 hrs) was seen on tissue culture plastic and Matrigel. The highest degree of differentiation (epithelioid appearance, apical villi and junctional complexes) was seen in cells plated on dishes coated with collagen IV and extracellular matrix secreted by endothelial cells. Cells were epithelioid and polarized on those two substrates; they expressed fine finger-shaped villi and the highest degree of cell contact (in the form of junctions). Cells grown on Matrigel looked like fibroblasts and became deeply pigmented; however, the nature of the pigment remains to be determined. Collagen IV and ECM coated dishes, therefore, are most suitable for cultures of human PE cell line 0041 because they provide higher cell densities while retaining the differentiated state. This is the first report where an established pigmented epithelial cell line has been induced to become differentiated by use of extracellular matrices and extracellular matrix components.

Protective effect of Sida cordata leaf extract against CCl(4) induced acute liver toxicity in rats.

OBJECTIVE: To investigate the hepatoprotective potential of Sida cordata (Malvaceae) (S. cordata) in experimental rats to validate its traditional claim. METHODS: Wister albino rats were divided into 6 groups: Group I served as control; Group II served as hepatotoxic (CCl(4) treated) group; Group III, IV and V served as (100, 200 and 400 mg/kg b.w.) S. cordata leaf extract (SCLE) treated groups; Group VI served as positive control (Silymarin) treated group. Liver marker enzymes serum glutamate oxyloacetic transaminase, serum glutamic pyruvic transaminase, pancreatic enzymatic antioxidants superoxide dismutase (SOD), lipid peroxidation, catalase (CAT), reduced glutathione (GSH) were measured and compared along with histopathological studies. RESULTS: Obtained results show that the treatment with SCLE significantly (P<0.05-<0.001) and dose-dependently reduced CCl4 induced elevated serum level of hepatic enzymes. Furthermore, SCLE significantly (up to P<0.001) reduced the lipid peroxidation in the liver tissue and restored activities of defence antioxidant enzymes GSH, SOD and CAT towards normal levels, which was confirmed by the histopathological studies. CONCLUSIONS: The results of this study strongly indicate the protective effect of SCLE against CCl(4) induced acute liver toxicity in rats and thereby scientifically support its traditional use.

Evaluation of anti-fertility activity of Tabernaemontana divaricata (Linn) R.Br. leaves in rats.

The aim of the present study was to assess the anti-fertility activity of ethanolic extracts of Tabernaemontana divaricata (TD) leaves in oestrogenic activity models in immature female rats. Mature green leaves of TD were collected and authenticated. Extractions of the dried leaves were carried out with ethanol in a Soxhlet's apparatus. For oestrogenic activity, the extracts were administered orally once daily at a dose of 200 and 400 mg kg(-1), and the activity was compared with the standard drug ethinyl oestradiol (0.02 mg). The extracts caused significant increase in uterine weight compared to the control. The ethanolic extract exhibited oestrogenic activity. The histological study of epithelium tissues with the 400 mg of TD extract-treated animals showed increases in the height of the luminal epithelium and loose edematous stroma when compared with the 200 mg of TD extract-treated group of animals. However, this was better than the control group of animals. Enhanced uterine weight and increase in the height of luminal epithelium and histological characteristics suggest that TD extract may be useful in anti-fertility therapy.