Characterization of Botulinum Neurotoxin Type A Neutralizing Monoclonal Antibodies and Influence of Their Half-Lives on Therapeutic Activity

Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent), intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab')2, Fab', scFv), chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs). F(ab')2 fragments, native or treated with polyethyleneglycol (PEG), were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17), recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination

Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine

Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90–120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined

Analysis of Ground Water Fluoride Content and its Association with Prevalence of Fluorosis in Zarand/Kerman: (Using GIS)

Abstract: Statement of Problem: The concentration of fluoride in water is usually higher in areas around the coal mines. Zarand region in the south-east of Iran is known for its coal mines. Some studies have shown the high prevalence of fluorosis and some studies reported high levels of fluoride in the region. Objectives: This study aimed to use Geographic Information System (GIS) to assess the relationship between water fluoride content and the prevalence of fluorosis and its spatial distribution in Zarand region. Materials and Methods: This cross-sectional study aimed to recruit 550 people aged 7-40 years in Zarand. Dental examination for fluorosis was conducted based on the Dean’s Index. The level of fluoride in the water was determined in samples of water taken from 35 areas. Information on fluorosis and fluoride content was mapped on GIS. Results: Most participants lived in rural areas (87.25%) and had an educational status of high school level (66%). About 23% of the examined people had normal teeth, 10% had severe and 67% had mild to moderate fluorosis. Distribution of severe fluorosis was higher in areas with higher levels of fluoride in the water according to GIS map. Conclusions: GIS map clearly showed a positive relationship between the prevalence and severity of fluorosis with the level of fluoride in water in Zarand. The GIS analysis may be useful in the analysis of other oral conditions

A Comparison of Differently Synthesized Gold-coated Magnetic Nanoparticles as 'Dispersible Electrodes'

Gold-coated magnetic nanoparticles (Au@MNPs) have attracted significant interest in electrochemistry in recent years. This is especially the case with their application as dispersible electrodes where modified Au@MNPs are dispersed into a solution, selectively bind to the analyte of interest and are then brought to an electrode via application of a magnetic field for measurement. This paper characterizes four types of Au@MNPs with different sizes, shapes, and method of synthesis as dispersible electrodes. The Au@MNPs were characterized by transmission electron microscopy and X-ray photoelectron spectroscopy and scanning electron microscopy. In addition, the electrochemical behaviour of Au@MNPs was investigated using cyclic voltammetry. The four sorts of Au@MNPs were evaluated with regards to the three main features required in the dispersible electrodes approach, well-defined morphology, well-defined electrochemistry and fast response to a magnetic field. The Cubic-Au@MNPs, which presents the simplest synthetic route, showed the best electrochemical stability and performance, responding quickly to a magnet and had a well defined shape

Analysis of Ground Water Fluoride Content and its Association with Prevalence of Fluorosis in Zarand/Kerman: (Using GIS)

STATEMENT OF PROBLEM: The concentration of fluoride in water is usually higher in areas around the coal mines. Zarand region in the south-east of Iran is known for its coal mines. Some studies have shown the high prevalence of fluorosis and some studies reported high levels of fluoride in the region. OBJECTIVES: This study aimed to use Geographic Information System (GIS) to assess the relationship between water fluoride content and the prevalence of fluorosis and its spatial distribution in Zarand region. MATERIALS AND METHODS: This cross-sectional study aimed to recruit 550 people aged 7-40 years in Zarand. Dental examination for fluorosis was conducted based on the Dean's Index. The level of fluoride in the water was determined in samples of water taken from 35 areas. Information on fluorosis and fluoride content was mapped on GIS. RESULTS: Most participants lived in rural areas (87.25%) and had an educational status of high school level (66%). About 23% of the examined people had normal teeth, 10% had severe and 67% had mild to moderate fluorosis. Distribution of severe fluorosis was higher in areas with higher levels of fluoride in the water according to GIS map. CONCLUSIONS: GIS map clearly showed a positive relationship between the prevalence and severity of fluorosis with the level of fluoride in water in Zarand. The GIS analysis may be useful in the analysis of other oral conditions

Analysis of Ground Water Fluoride Content and its Association with Prevalence of Fluorosis in Zarand/Kerman: (Using GIS)

STATEMENT OF PROBLEMThe concentration of fluoride in water is usually higher in areas around the coal mines. Zarand region in the south-east of Iran is known for its coal mines. Some studies have shown the high prevalence of fluorosis and some studies reported high levels of fluoride in the region. OBJECTIVESThis study aimed to use Geographic Information System (GIS) to assess the relationship between water fluoride content and the prevalence of fluorosis and its spatial distribution in Zarand region. MATERIALS AND METHODSThis cross-sectional study aimed to recruit 550 people aged 7-40 years in Zarand. Dental examination for fluorosis was conducted based on the Dean's Index. The level of fluoride in the water was determined in samples of water taken from 35 areas. Information on fluorosis and fluoride content was mapped on GIS. RESULTSMost participants lived in rural areas (87.25%) and had an educational status of high school level (66%). About 23% of the examined people had normal teeth, 10% had severe and 67% had mild to moderate fluorosis. Distribution of severe fluorosis was higher in areas with higher levels of fluoride in the water according to GIS map. CONCLUSIONSGIS map clearly showed a positive relationship between the prevalence and severity of fluorosis with the level of fluoride in water in Zarand. The GIS analysis may be useful in the analysis of other oral conditions

Connecting electrodes with light: One wire, many electrodes

The requirement of a wire to each electrode is central to the design of any electronic device but can also be a major restriction. For example it entails space restrictions and rigid device architecture in multi-electrode devices. The finite space that is taken up by the array of electrical terminals and conductive pads also severely limits the achievable density of electrodes in the device. Here it is shown that a travelling light pointer can be used to form transient electrical connections anywhere on a monolithic semiconductor electrode that is fitted with a single peripheral electrical terminal. This is achieved using hydrogen terminated silicon electrodes that are modified with well-defined organic monolayers. It is shown that electrochemical information can be either read from or written onto these surfaces. Using this concept it is possible to form devices that are equivalent to a conventional electrode array but that do not require a predetermined architecture, and where each element of the array is temporally "connected" using light stimulus; a step change in capability for electrochemistry

The potential role of heat shock proteins in cardiovascular disease: evidence from in vitro and in vivo studies

The heat shock proteins (HSPs) are highly conserved families of proteins expressed by a number of cell types following exposure to stressful environmental conditions. These conditions include several known risk factors for cardiovascular disease. A number of the HSPs have been shown to be molecular chaperones that are involved in the refolding of other damaged protein molecules. Over the past two decades there has been an increasing interest in the relationship between HSPs and cardiovascular disease, and particularly whether an autoimmune response may be implicated. The fact that microorganisms also produce HSPs, and that these are homologous to human HSPs has given rise to concept of molecular mimicry. While most of the past studies have focused on HSP 65 and 70, there has been recent interest and investigations of the possible role of the smaller HSPs, such as HSP27, in atherogenesis. Furthermore, the possibility that autoimmunity may be mediating the deleterious effects of HSPs has led some investigators to explore tolerization as a potential therapeutic approach

DNA-hybridisation detection on Si(100) surfaces using lightactivated electrochemistry: a comparative study between bovine serum albumin and hexaethylene glycol as antifouling layers

Light can be used to spatially resolve electrochemical measurements on a semiconductor electrode. This phenomenon has been explored to detect DNA hybridization with light-addressable potentiometric sensors and, more recently, with light-addressable amperometric sensors based on organic-monolayer-protected Si(100). Here, a contribution to the field is presented by comparing sensing performances when bovine serum albumin (BSA) and hexaethylene glycol (OEG₆) are employed as antifouling layers that resist nonspecific adsorption to the DNA-modified interface on Si(100) devices. What is observed is that both sensors based on BSA or OEG₆ initially allow electrochemical distinction among complementary, noncomplementary, and mismatched DNA targets. However, only surfaces based on OEG₆ can sustain electroactivity over time. Our results suggest that this relates to accelerated SiO_<i>x</i> formation occasioned by BSA proteins adsorbing on monolayer-protected Si(100) surfaces. Therefore, DNA biosensors were analytically explored on low-doped Si(100) electrodes modified on the molecular level with OEG₆ as an antifouling layer. First, light-activated electrochemical responses were recorded over a range of complementary DNA target concentrations. A linear semilog relation was obtained from 1.0 × 10^–11 to 1.0 × 10^–6 mol L^–1 with a correlation coefficient of 0.942. Then, measurements with three independent surfaces indicated a relative standard deviation of 4.5%. Finally, selectivity tests were successfully performed in complex samples consisting of a cocktail mixture of four different DNA sequences. Together, these results indicate that reliable and stable light-activated amperometric DNA sensors can be achieved on Si(100) by employing OEG₆ as an antifouling layer