1.
Vicente Carrillo, Alejandro, 1989-
(author)
Sperm Membrane Channels, Receptors and Kinematics : Using boar spermatozoa for drug toxicity screening
2016
Doctoral thesis (other academic/artistic) abstract
Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.
2.
Ekstrand, Carl, et al.
(author)
Plasma concentration-dependent suppression of endogenous hydrocortisone in the horse after intramuscular administration of dexamethasone-21-isonicotinate
2015
In: Journal of Veterinary Pharmacology and Therapeutics. - : Wiley. - 0140-7783 .- 1365-2885. ; 38:3, s. 235-242
Journal article (peer-reviewed) abstract
Detection times and screening limits (SL) are methods used to ensure that the performance of horses in equestrian sports is not altered by drugs. Drug concentration-response relationship and knowledge of concentration-time profiles in both plasma and urine are required. In this study, dexamethasone plasma and urine concentration-time profiles were investigated. Endogenous hydrocortisone plasma concentrations and their relationship to dexamethasone plasma concentrations were also explored. A single dose of dexamethasone-21-isonicotinate suspension (0.03mg/kg) was administered intramuscularly to six horses. Plasma was analysed for dexamethasone and hydrocortisone and urine for dexamethasone, using UPLC-MS/MS. Dexamethasone was quantifiable in plasma for 8.3 +/- 2.9days (LLOQ: 0.025g/L) and in urine for 9.8 +/- 3.1days (LLOQ: 0.15g/L). Maximum observed dexamethasone concentration in plasma was 0.61 +/- 0.12g/L and in urine 4.2 +/- 0.9g/L. Terminal plasma half-life was 38.7 +/- 19h. Hydrocortisone was significantly suppressed for 140h. The plasma half-life of hydrocortisone was 2.7 +/- 1.3h. Dexamethasone potency, efficacy and sigmoidicity factor for hydrocortisone suppression were 0.06 +/- 0.04g/L, 0.95 +/- 0.04 and 6.2 +/- 4.6, respectively. Hydrocortisone suppression relates to the plasma concentration of dexamethasone. Thus, determination of irrelevant plasma concentrations and SL is possible. Future research will determine whether hydrocortisone suppression can be used as a biomarker of the clinical effect of dexamethasone.
3.
Hansson, Elisabeth, 1955, et al.
(author)
Bupivacaine in combination with sildenafil (Viagra) and vitamin D3 have anti-inflammatory effects in osteoarthritic chondrocytes
2021
In: Current Research in Pharmacology and Drug Discovery. - : Elsevier BV. - 2590-2571. ; 2
Journal article (peer-reviewed) abstract
Aims: To treat osteoarthritic chondrocytes and thereby reduce the inflammation with a drug combination that primarily affects 5-HT- and ATP-evoked Ca2+ signaling. In osteoarthritic chondrocytes, Ca2+ signaling is elevated, resulting in increased production of ATP and inflammatory mediators. The expression of TLR4 and Na+/K+-ATPase was used to evaluate the inflammatory status of the cells. Main methods: Equine chondrocytes were collected from joints with mild structural osteoarthritic changes and cultured in monolayers. The cells were treated with a combination of bupivacaine (1 pM) and sildenafil (1 μM) in combination with vitamin D3 (100 nM). A high-throughput screening system, the Flexstation 3 microplate reader, was used to measure intra- and extracellular Ca2+ signaling after exposure to 5-HT, glutamate, or ATP. Expression of inflammatory receptors was assessed by Western blotting. Key findings: Drug treatment substantially reduced 5-HT- and ATP-evoked intracellular Ca2+ release and TLR4 expression compared to those in untreated chondrocytes. The combination of sildenafil, vitamin D3 together with metformin, as the ability to take up glucose is limited, increased Na+/K+-ATPase expression. Significance: The combination of these three therapeutic substances at concentrations much lower than usually used, reduced expression of the inflammatory receptor TLR4 and increased the cell membrane enzyme Na+/K+-ATPase, which regulates cell volume and reduces increased intracellular Ca2+ concentrations. These remarkable results indicate that this drug combination has disease-modifying osteoarthritis drug (DMOAD) properties and may be a new clinical therapy for osteoarthritis (OA). © 2021 The Authors
4.
Stroikin, Yuri, et al.
(author)
Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes
2008
In: Age (Omaha). - : Springer. - 0161-9152 .- 1574-4647. ; 30:1, s. 31-42
Journal article (peer-reviewed) abstract
Cellular ageing is associated with accumulation of undegradable intralysosomal material, called lipofuscin. In order to accelerate the lipofuscin-accumulation, confluent, growth arrested human fibroblasts were cultured under hyperoxic conditions. To provide a better insight into the effects of lipofuscin on cellular functions, we compared lysosomal stability in control and lipofuscin-loaded human fibroblasts under conditions of lysosome-targeted stress induced by exposure to either the lysosomotropic detergent MSDH or the redox-cycling quinone naphthazarin. We show that lysosomal damage, assessed by acridine-orange relocation, translocation of cathepsin D to the cytosol, and alkalinization of lysosomes is more pronounced in control than in lipofuscin-loaded fibroblasts. Finding that lysosomal integrity was less affected or even preserved in case of lipofuscin-loaded cells enables us to suggest that lipofuscin exerts lysosome-stabilizing properties.
5.
Held, Felix, et al.
(author)
Modelling of oscillatory cortisol response in horses using a Bayesian population approach for evaluation of dexamethasone suppression test protocols
2019
In: Journal of Pharmacokinetics and Pharmacodynamics. - : Springer Science and Business Media LLC. - 1567-567X .- 1573-8744. ; 46:1, s. 75-87
Journal article (peer-reviewed) abstract
Cortisol is a steroid hormone relevant to immune function in horses and other species and shows a circadian rhythm. The glucocorticoid dexamethasone suppresses cortisol in horses. Pituitary pars intermedia dysfunction (PPID) is a disease in which the cortisol suppression mechanism through dexamethasone is challenged. Overnight dexamethasone suppression test (DST) protocols are used to test the functioning of this mechanism and to establish a diagnosis for PPID. However, existing DST protocols have been recognized to perform poorly in previous experimental studies, often indicating presence of PPID in healthy horses. This study uses a pharmacokinetic/pharmacodynamic (PK/PD) modelling approach to analyse the oscillatory cortisol response and its interaction with dexamethasone. Two existing DST protocols were then scrutinized using model simulations with particular focus on their ability to avoid false positive outcomes. Using a Bayesian population approach allowed for quantification of uncertainty and enabled predictions for a broader population of horses than the underlying sample. Dose selection and sampling time point were both determined to have large influence on the number of false positives. Advice on pitfalls in test protocols and directions for possible improvement of DST protocols were given. The presented methodology is also easily extended to other clinical test protocols.
6.
Lundälv, Jörgen, 1966
(author)
Bioterrorism och media
2004
Book (other academic/artistic) abstract
Attacker med biologiska och kemiska preparat är ett nytt hot mot institutioner i världen bl.a massmedia. I USA har ett antal medieföretag attackerats med brev som visat sig innehålla mjältbrand. I Sverige utsätts medier för hot och risker med jämna mellanrum. Denna guide om säkerhet och beredskap ger ny kunskap om vad bioterrorism innebär och vilka hotbilder som finns mot medieföretag i Sverige. Den vänder sig också till informationsstrateger vid myndigheter och företag liksom till hälso- och sjukvårdspersonal med intresse för epidemiologiska frågor. Guiden inleds med företal av Gorm Albrechtsen, f.d. chefredaktör vid Herning Folkeblad i Danmark som utsatts för misstänkta pulverbrev samt av Åke Sellström, avdelningschef vid Totalförsvarets forskningsinstitut (FOI) och expert på biologiska och kemiska vapen.
7.
Hasan, Badrul, 1984-, et al.
(author)
The Gull (Chroicocephalus brunnicephalus) as an Environmental Bioindicator and Reservoir for Antibiotic Resistance on the Coastlines of the Bay of Bengal
2014
In: Microbial Drug Resistance. - : Mary Ann Liebert Inc. - 1076-6294 .- 1931-8448. ; 20:5, s. 466-471
Journal article (peer-reviewed) abstract
The presence and frequency of multiresistant bacteria in wild birds act as indicators of the environmental contamination of antibiotic resistance. To explore the rate of contamination mediated by Escherichia coli, 150 fecal samples from the brown-headed gull (Chroicocephalus brunnicephalus) and 8 water samples from the Bay of Bengal area were collected, cultured, and tested for antibiotic susceptibility. Special attention was paid to extended-spectrum beta-lactamase (ESBL)-producing isolates, which were further characterized genetically. Antibiotic resistance was found in 42.3% (36/85) of the E. coli isolates and multidrug resistance in 11.8%. Isolates from the area with a higher human activity were more resistant than those from an area with a lower level of activity. Most frequent was resistance to ampicillin (29.4%), followed by trimethoprim-sulfamethoxazole (24.7%) and quinolones (22.4%). Carriage of ESBL-producing E. coli was relatively high (17.3%) in the gulls, whereas no ESBL producers were found in the water. All ESBL-producing E. coli isolates, but one, carried blaCTX-M-15 or blaCTX-M-15-like genes. A blaCTX-M-14-like enzyme was found as an exception. Gulls from two different colonies shared E. coli clones and harbored the clinically relevant sequence types ST10, ST48, and ST131. The high frequency of antibiotic resistance and ESBL production among E. coli isolates from gulls indicates that the environmental contamination of antibiotic resistance has already gone far on the coastlines of the Bay of Bengal. Considering the limited control over the antibiotic consumption and waste from human activities in Bangladesh, there is no easy solution in sight.
8.
9.
10.
Eriksson, Staffan, et al.
(author)
Differences in cytosolic and mitochondrial 5 '-nucleotidase and deoxynucleoside kinase activities in Sprague-Dawley rat and CD-1 mouse tissues: Implication for the toxicity of nucleoside analogs in animal models
2010
In: Toxicology. - : Elsevier BV. - 0300-483X .- 1879-3185. ; 267, s. 159-164
Journal article (peer-reviewed) abstract
Cytosolic and mitochondrial deoxynucleoside kinases (dNKs), as well as 5'deoxynucleotidases (5'-dNTs), control intracellular and intramitochondrial phosphorylation of natural nucleotides and nucleoside analogs used in antiviral and cancer chemotherapy. The balance in the activities of these two groups of enzymes to a large extent determines both the efficacy and side effects of these drugs. Because of the broad and overlapping substrate specificities of the nucleoside kinases and 5'-NTs, their tissue distribution and roles in the metabolism of both natural nucleosides and their analogs are still not fully elucidated. Here, the activity of dNKs: dCK and TK (TK1 and TK2) as well as 5'-dNTs: CN1, CN2 and dNT (dNT1 and dNT2) were determined in 14 different adult mouse and rat tissues. In most cases tissue activities of TK1, TK2 and dCK were 2-3-fold higher in the mouse, a similar pattern was found with CN1 and dNTs although with several exceptions, e.g., TK2 activities in muscle extracts from rats were 2-10-fold higher than in the mouse. Furthermore CN1 activities in hepatic, renal and adipose extracts were 2-3-fold higher in the rat. CN2 had higher levels in the testis, spleen, pancreas and diaphragm and lower level in the lung of mouse compared to rat tissues. The result suggests that a major difference in these activity profiles between mouse and rat may account for discrepancies in pharmacological response of the two animals to certain nucleoside compounds, and may help to improve the usefulness of animal models in future efforts of drug discovery. (C) 2009 Elsevier Ireland Ltd. All rights reserved.
