Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia¹. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar². Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells³. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase^4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.     

Expression,glycosylation and secretion of yeast acid phosphatase in hamster BHK cells

The gene PHO5 coding for one of the repressible acid phosphatases of the yeastSaccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human -actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular massM _r=62000, indicating substitution of the polypeptide moiety by 2–3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.Abbreviations APase acid phosphatase - PBS phosphate buffered saline - TBS Tris buffered saline - con A concanavalin A - TCA Tetracarpidium conophorum agglutinin     

Regulation of therapeutic apoptosis: a potential target in controlling hypertensive organ damage

Cell growth and survival are potential therapeutic targets for the control of complications associated with hypertension. In most cardiovascular disorders, cardiac fibroblasts and large-vessel smooth muscle cells can replicate and thus contribute to the disease. We propose that cardiovascular hyperplasia may be reversed via therapeutic apoptosis induction with drugs that are safe and already used in the clinic. We first reported that, irrespective of the drug class, those drugs that are able to induce regression of cardiovascular hypertrophy are also able to reverse cardiovascular hyperplasia via apoptosis. Drugs active in this regard include inhibitors of the renin-angiotensin system, calcium channel blockers, and beta-blockers. Moreover, the effects of these drugs on cell survival is not merely secondary to blood pressure reduction. Therapeutic apoptosis in the cardiovascular system of the spontaneously hypertensive rat is characterized by a rapid and transient onset following initiation of antihypertensive treatment. Herein, the induction and termination of therapeutic apoptosis during drug treatment of hypertension will be briefly reviewed and supported by novel data suggesting that reversal of cardiovascular hyperplasia is associated with reduced cell growth and a resistance to further induction of therapeutic apoptosis, as shown in spontaneously hypertensive rats receiving an intermittent regime of nifedipine therapy. We propose that the presence of a cell subpopulation with defective cell cycle regulation may determine organ susceptibility to undergo therapeutic apoptosis.     

Antigens of selected Acanthamoeba species detected with monoclonal antibodies

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.     

An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the attachment organelle of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus. Abbreviations CSS Corn stunt spiroplasma - SEM Scanning electron microscopy - TBS Tris-buffered saline - TEM Transmission electron microscopy     

Counties selecting public health priorities--a "bottom-up" approach (Croatian experience)

The subject of this paper is how to incorporate a multi-disciplinary and inter-sectored approach into development of public health policy and plans at the local (county) level in Croatia by educational program. Method used was the public health capacity building program "Health--Plan for it", which was developed with the aim to assist the counties to overcome recognized weaknesses and introduce more effective and efficient local public health practices. Two main instruments were used: Local Public Health Practice Performance Measures Instrument, and Basic Priority Rating System. This program has helped counties to asses population health needs in a participatory manner, to plan for health and, ultimately, assure provision of the right kind and quality of services (better tailored to population health needs). This program's benefits are going beyond and above the county level. It provides support for the Healthy Cities project locally, and facilitates changes in national policymaking body's mindset that a "one-size-fits-all" approach is sufficient.     

Immune cell phenotype and functional defects in Netherton syndrome

Background

Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete.

Results

We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17?years, and healthy age-matched controls. The proportion of B cells (CD19⁺) and naïve B cells (CD27^?, IgD⁺) were high while memory B cells (CD27⁺) and switched memory B cells (CD27⁺IgM^?IgD^?), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21^low, CD38l^ow) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4⁺ T cells was reduced significantly and the proportion of CD8⁺ T central memory significantly elevated. An increased proportion of CD57⁺ CD8⁺ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16⁺ and CD27^? NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity.The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L⁺ T cells, naïve CD4⁺ and CD27⁺ CD8⁺ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8⁺ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied.

Conclusions

This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

     

Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes

Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER). We found that the fusion protein showed higher levels of expression when compared to the fusion proteins using rice amylase 3D (RAmy3D) or CTB native signal sequence only. The CTB-cEDIII fusion protein was evaluated as an oral dengue vaccine candidate in mice. Serotype specific systemic IgG antibodies and specific IgA response in feces were detected and furthermore, T cell proliferation and high frequency antibody-secreting B cells were detected in the spleen. These results suggest the possible use of plant-based dengue tetravalent vaccine targeted to the mucosal immune system for induction of systemic and mucosal immune responses to DENV infection.