The treatment lottery of chronic back pain? A case series at a multidisciplinary pain centre.

OBJECTIVES: Despite the number of people affected by chronic back pain, and the many available treatment options, even the best modalities provide limited pain reduction on a group level, often without simultaneous improvements in functioning or health-related quality of life. The objective was to provide an overview of the treatment of chronic back pain in clinical practice at a multidisciplinary pain centre, and to study patient and pain characteristics in different treatment groups. METHODS: 104 chronic back pain patients (primary ICD-10-SE-diagnosis M53.0-M54.9 excluding M54.1 and M54.3), referred to the Pain and Rehabilitation Centre, University Hospital, Linköping in 2015, were studied using data from the Swedish Quality Registry for Pain Rehabilitation, self-reported medication data, and a retrospective medical record review. RESULTS: The following treatment groups were identified: rehabilitation (n=21), analgesics (n=33), invasive intervention (n=14), and no treatment (n=35). Significant differences between groups were found with regards to age, sick leave, education level, persisting pain duration, punishing responses by significant other, previous invasive intervention, receiving sub-clinic, physician speciality and referring care level. CONCLUSIONS: Overall, patient demographics were associated with treatment strategy to a higher degree than patient-reported outcome measures. Moreover, physician speciality and organisational factors seemed to play a role in treatment choice.

Increased apoptosis, reduced Wnt/ß-catenin signaling, and altered tail development in zebrafish embryos exposed to a human-relevant chemical mixture.

A wide variety of anthropogenic chemicals is detected in humans and wildlife and the health effects of various chemical exposures are not well understood. Early life stages are generally the most susceptible to chemical disruption and developmental exposure can cause disease in adulthood, but the mechanistic understanding of such effects is poor. Within the EU project EDC-MixRisk, a chemical mixture (Mixture G) was identified in the Swedish pregnancy cohort SELMA by the inverse association between levels in women at around gestational week ten with birth weight of their children. This mixture was composed of mono-ethyl phthalate, mono-butyl phthalate, mono-benzyl phthalate, mono-ethylhexyl phthalate, mono-isononyl phthalate, triclosan, perfluorohexane sulfonate, perfluorooctanoic acid, and perfluorooctane sulfonate. In a series of experimental studies, we characterized effects of Mixture G on early development in zebrafish models. Here, we studied apoptosis and Wnt/ß-catenin signaling which are two evolutionarily conserved signaling pathways of crucial importance during development. We determined effects on apoptosis by measuring TUNEL staining, caspase-3 activity, and acridine orange staining in wildtype zebrafish embryos, while Wnt/ß-catenin signaling was assayed using a transgenic line expressing an EGFP reporter at ß-catenin-regulated promoters. We found that Mixture G increased apoptosis, suppressed Wnt/ß-catenin signaling in the caudal fin, and altered the shape of the caudal fin at water concentrations only 20-100 times higher than the geometric mean serum concentration in the human cohort. These findings call for awareness that pollutant mixtures like mixture G may interfere with a variety of developmental processes, possibly resulting in adverse health effects.

Bisphenol AF and Bisphenol F Induce Similar Feminizing Effects in Chicken Embryo Testis as Bisphenol A.

The plastic component bisphenol A (BPA) impairs reproductive organ development in various experimental animal species. In birds, effects are similar to those caused by other xenoestrogens. Because of its endocrine disrupting activity, BPA is being substituted with other bisphenols in many applications. Using the chicken embryo model, we explored whether the BPA alternatives bisphenol AF (BPAF), bisphenol F (BPF), and bisphenol S (BPS) can induce effects on reproductive organ development similar to those induced by BPA. Embryos were exposed in ovo from embryonic day 4 (E4) to vehicle, BPAF at 2.1, 21, 210, and 520 nmol/g egg, or to BPA, BPF, or BPS at 210 nmol/g egg and were dissected on embryonic day 19. Similar to BPA, BPAF and BPF induced testis feminization, manifested as eg testis-size asymmetry and ovarian-like cortex in the left testis. In the BPS-group, too few males were alive on day 19 to evaluate any effects on testis development. We found no effects by any treatment on ovaries or Müllerian ducts. BPAF and BPS increased the gallbladder-somatic index and BPAF, BPF and BPS caused increased embryo mortality. The overall lowest-observed-adverse-effect level for BPAF was 210 nmol/g egg based on increased mortality, increased gallbladder-somatic index, and various signs of testis feminization. This study demonstrates that the BPA replacements BPAF, BPF, and BPS are embryotoxic and suggests that BPAF is at least as potent as BPA in inducing estrogen-like effects in chicken embryos. Our results support the notion that these bisphenols are not safe alternatives to BPA.

A suggested bisphenol A metabolite (MBP) interfered with reproductive organ development in the chicken embryo while a human-relevant mixture of phthalate monoesters had no such effects.

Bisphenol A (BPA) and phthalate diesters are ubiquitous environmental contaminants. While these compounds have been reported as reproductive toxicants, their effects may partially be attributed to metabolites. The aim of this study was to examine reproductive organ development in chicken embryos exposed to the BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP; 100 µg/g egg) or a human-relevant mixture of 4 phthalate monoesters (85 µg/g egg). The mixture was designed within the EU project EDC-MixRisk based upon a negative association with anogenital distance in boys at 21 months of age in a Swedish pregnancy cohort. Chicken embryos were exposed in ovo from an initial stage of gonad differentiation (embryonic day 4) and dissected two days prior to anticipated hatching (embryonic day 19). No discernible effects were noted on reproductive organs in embryos exposed to the mixture. MBP-treated males exhibited retention of Müllerian ducts and feminization of the left testicle, while MBP-administered females displayed a diminished the left ovary. In the left testicle of MBP-treated males, mRNA expression of female-associated genes was upregulated while the testicular marker gene SOX9 was downregulated, corroborating a feminizing effect by MBP. Our results demonstrate that MBP, but not the phthalate monoester mixture, disrupts both male and female reproductive organ development in an avian embryo model.

Developmental exposure to a human relevant mixture of endocrine disruptors alters metabolism and adipogenesis in zebrafish (Danio rerio).

Exposure to endocrine disrupting chemicals has been suggested to contribute to the ongoing globally increasing obesity trend. The complex chemical mixtures that humans and wildlife are exposed to include a number of compounds that may have obesogenic properties. In this study we examined a mixture consisting of phthalate-monoesters, triclosan, and perfluorinated compounds. The mixture was designed within the EDC-MixRisk project based on serum levels of the compounds in pregnant women of a Swedish mother-child cohort. The compounds were negatively associated with birth weight of the children. We assessed whether developmental exposure to this mixture in combination with a calorie-rich diet affected metabolic rate, blood lipids, adipogenesis and lipid storage, and the whole-body level of neutral lipids in zebrafish (Danio rerio). Wildtype zebrafish were exposed to the mixture from 3 h post fertilization to 5, 14 or 17 days post fertilization (dpf) at water concentrations corresponding to 1, 10, 20, or 100 times the geometrical mean of the serum concentration (hsc) in the women. Exposure to the mixture at 20 times hsc lowered metabolic rate at 2-5 dpf, and increased the number of adipocytes and the amount of visceral adipose tissue at 14 and 17 dpf respectively. Also, mRNA expression of fatty acid binding protein 11a was increased at 17 dpf by 10 and 20 times hsc of the mixture. This study shows that a human-relevant mixture of environmental pollutants affects metabolic rate, adipogenesis and lipid storage in young zebrafish fed a calorie-rich diet, thus demonstrating its potential to disrupt metabolism.

Developmental exposure to a mixture of perfluoroalkyl acids (PFAAs) affects the thyroid hormone system and the bursa of Fabricius in the chicken.

Perfluoroalkyl acids (PFAAs) are ubiquitous environmental contaminants and eggs and nestlings of raptors and fish-eating birds often contain high levels of PFAAs. We studied developmental effects of a mixture of ten PFAAs by exposing chicken embryos to 0.5 or 3 µg/g egg of each compound in the mixture. Histological changes of the thyroid gland were noted at both doses and increased expression of mRNA coding for type III deiodinase was found at 0.5 µg/g egg. Serum concentrations of the free fraction of thyroid hormones (T3 and T4) were reduced by the PFAA mixture at 3 µg/g egg, which is in line with a decreased synthesis and increased turnover of thyroid hormones as indicated by our histological findings and the decreased mRNA expression of type III deiodinase. The relative weight of the bursa of Fabricius increased at a dose of 3 µg/g egg in females. The bursa is the site of B-cell development in birds and is crucial for the avian adaptive immune system. Analysis of plasma and liver concentrations of the mixture components showed differences depending on chain length and functional group. Our results highlight the vulnerability of the thyroid hormone and immune systems to PFAAs.

[Medical simulation training ­ an overview of the evidence]. / Simuleringsträning ger ökad kunskap och bättre färdigheter - Men osäkerhet råder avseende klinisk nytta då många studier brister i evidens.

Medical simulation training - an overview of the evidence Medical simulation training has become an important model for training of technical and non-technical clinical skills, aiming at preventing avoidable mistakes. The evidence for effects of simulation training is increasing, and several systematic reviews on the effect of medical simulation training have been published. This article summarizes the evidence for medical simulation training based on systematic reviews of medical simulation published in the last three years. There is a consistent finding in all systematic reviews that simulation training has a positive effect on learning and skills transfer, and small positive effects on patient-related outcomes. However, there is considerable uncertainty about the strength of evidence of this research, and all systematic reviews reported serious weaknesses in research methods of the included studies. We hope that the newly published guidelines for study design and reporting of medical simulation research can help create a stronger evidence base.

Effects of selective and combined activation of estrogen receptor α and ß on reproductive organ development and sexual behaviour in Japanese quail (Coturnix japonica).

Excess estrogen exposure of avian embryos perturbs reproductive organ development in both sexes and demasculinizes the reproductive behaviors of adult males. We have previously shown that these characteristic effects on the reproductive organs also can be induced by exposure of Japanese quail (Coturnix japonica) embryos to selective agonists of estrogen receptor alpha (ERα). In contrast, the male copulatory behavior is only weakly affected by developmental exposure to an ERα agonist. To further elucidate the respective roles of ERα and ERß in estrogen-induced disruption of sexual differentiation, we exposed Japanese quail embryos in ovo to the selective ERα agonist 16α-lactone-estradiol (16αLE2), the selective ERß agonist WAY-200070, or both substances in combination. The ERα agonist feminized the testes in male embryos and reduced cloacal gland size in adult males. Furthermore, anomalous retention and malformations of the Müllerian ducts/oviducts were seen in embryos and juveniles of both sexes. The ERß agonist did not induce any of these effects and did not influence the action of the ERα agonist. Male copulatory behavior was not affected by embryonic exposure to either the ERα- or the ERß-selective agonist but was slightly suppressed by treatment with the two compounds combined. Our results suggest that the reproductive organs become sexually differentiated consequent to activation of ERα by endogenous estrogens; excessive activation of ERα, but not ERß, during embryonic development may disrupt this process. Our results also suggest that the demasculinizing effect of estrogens on male copulatory behavior is only partly mediated by ERα and ERß, and may rather involve other estrogen-responsive pathways.

Differential Effect of APOE Alleles on Brain Glucose Metabolism in Targeted Replacement Mice: An [18F]FDG-µPET Study.

BACKGROUND: The Apolipoprotein E (ApoE) alleles ɛ2, ɛ3, and ɛ4 are known to differentially modulate cerebral glucose metabolism and the risk for Alzheimer's disease (AD) via both amyloid-ß (Aß)-dependent and independent mechanisms. OBJECTIVE: We investigated the influence of ApoE on cerebral glucose metabolism in humanized APOE Targeted Replacement (TR) mice at ages that precede the comparison of Aß parenchymal deposits in APOE4-TR mice. METHODS: Fludeoxyglucose ([18F]FDG) positron emission tomography (PET) measures were performed longitudinally in homozygous APOE-TR mice (APOE2, APOE3, APOE4; n = 10 for each group) at 3, 5, 11, and 15 months. Results were quantified using standard uptake values and analyzed statistically using a linear mixed effects model. Levels of the Aß40 and Aß42 peptides were quantified ex vivo using enzyme-linked immunosorbent assay (ELISA) at 15 months in the same animals. RESULTS: APOE2 mice (versus APOE3) showed a significant increase in glucose metabolism starting at 6 months, peaking at 9 months. No evidence of hypometabolism was apparent in any region or time point for APOE4 mice, which instead displayed a hypermetabolism at 15 months. Whole brain soluble Aß40 and Aß42 levels were not significantly different between genotypes at 15 months. CONCLUSIONS: Introduction of human APOE alleles ɛ2 and ɛ4 is sufficient to produce alterations in brain glucose metabolism in comparison to the control allele ɛ3, without a concomitant alteration in Aß40 and Aß42 levels. These results suggest novel Aß-independent metabolic phenotypes conferred by ɛ2 and ɛ4 alleles and have important implications for preclinical studies using TR-mice.

Sex-dependent expression of anti-Müllerian hormone (amh) and amh receptor 2 during sex organ differentiation and characterization of the Müllerian duct development in Xenopus tropicalis.

Amphibian gonadal differentiation involves the action of sex steroids. Recent research indicates that the anti-Müllerian hormone (AMH) is involved in testicular development in some lower vertebrate species. For amphibians there is a lack of data on ontogenetic expression of the AMH receptor AMHR2/amhr2 and of progesterone receptors (PGRS/pgrs). Here we expand the knowledge on amphibian sex differentiation by characterizing ontogenetic mRNA levels of amh, amhr2, intracellular and membrane pgrs (ipgr and mpgr beta) and cytochrome P450 19a1 (cyp19a1) (ovarian marker) in the urogenital complex of the model species Xenopus (Silurana) tropicalis. Furthermore, we characterized the ontogenetic development of the Müllerian ducts (precursors of the female reproductive tract) histologically. The developmental period investigated spanned from beginning of gonadal differentiation, Nieuwkoop and Faber (NF) stage 51, to 4weeks post-metamorphosis. The Müllerian ducts were first observed at NF 64 in both sexes. Male-enhanced amh mRNA levels from NF 53/54 to 6days post-metamorphosis and female-enhanced cyp19a1 levels from NF 53 to 4weeks post-metamorphosis were noted. The sexually dimorphic mRNA level profile was more distinct for amh than for cyp19a1. The pgrs mRNA levels increased over the studied period and showed no sex differences. At later developmental stages, the amhr2 mRNA level was increased in putative females compared with males. Our findings suggest that AMH has a role in gonadal differentiation in X. tropicalis. We propose relative gonadal amh mRNA level as a testicular marker during early gonadal development in amphibians.

NgR1: A Tunable Sensor Regulating Memory Formation, Synaptic, and Dendritic Plasticity.

Nogo receptor 1 (NgR1) is expressed in forebrain neurons and mediates nerve growth inhibition in response to Nogo and other ligands. Neuronal activity downregulates NgR1 and the inability to downregulate NgR1 impairs long-term memory. We investigated behavior in a serial behavioral paradigm in mice that overexpress or lack NgR1, finding impaired locomotor behavior and recognition memory in mice lacking NgR1 and impaired sequential spatial learning in NgR1 overexpressing mice. We also investigated a role for NgR1 in drug-mediated sensitization and found that repeated cocaine exposure caused stronger locomotor responses but limited development of stereotypies in NgR1 overexpressing mice. This suggests that NgR1-regulated synaptic plasticity is needed to develop stereotypies. Ex vivo magnetic resonance imaging and diffusion tensor imaging analyses of NgR1 overexpressing brains did not reveal any major alterations. NgR1 overexpression resulted in significantly reduced density of mature spines and dendritic complexity. NgR1 overexpression also altered cocaine-induced effects on spine plasticity. Our results show that NgR1 is a negative regulator of both structural synaptic plasticity and dendritic complexity in a brain region-specific manner, and highlight anterior cingulate cortex as a key area for memory-related plasticity.

Toxicity and cytochrome P450 1A mRNA induction by 6-formylindolo[3,2-b]carbazole (FICZ) in chicken and Japanese quail embryos.

The tryptophan derivative formylindolo[3,2-b]carbazole (FICZ) binds with high ligand affinity to the aryl hydrocarbon receptor (AHR) and is readily degraded by AHR-regulated cytochrome P450 family 1 (CYP1) enzymes. Whether in vivo exposure to FICZ can result in toxic effects has not been examined and the main objective of this study was to determine if FICZ is embryotoxic in birds. We examined toxicity and CYP1 mRNA induction of FICZ in embryos from chicken (Gallus domesticus) and Japanese quail (Coturnix japonica) exposed to FICZ (2-200µgkg(-1)) by yolk and air sac injections. FICZ caused liver toxicity, embryo mortality, and CYP1A4 and CYP1A5 induction in both species with similar potency. This is in stark contrast to the very large difference in sensitivity of these species to halogenated AHR agonists. We also exposed chicken embryos to a low dose of FICZ (4µgkg(-1)) in combination with a CYP inhibitor, ketoconazole (KCZ). The mixture of FICZ and KCZ was lethal while FICZ alone had no effect at 4µgkg(-1). Furthermore, mixed exposure to FICZ and KCZ caused stronger and more long-lasting hepatic CYP1A4 induction than exposure to each compound alone. These findings indicate reduced biotransformation of FICZ by co-treatment with KCZ as a cause for the enhanced effects although additive AHR activation is also possible. To conclude, FICZ is toxic to bird embryos and it seems reasonable that the toxicity by FICZ involves AHR activation. However, the molecular targets and biological events leading to hepatic damage and mortality are unknown.

Metabolic Profiling of Chicken Embryos Exposed to Perfluorooctanoic Acid (PFOA) and Agonists to Peroxisome Proliferator-Activated Receptors.

Untargeted metabolic profiling of body fluids in experimental animals and humans exposed to chemicals may reveal early signs of toxicity and indicate toxicity pathways. Avian embryos develop separately from their mothers, which gives unique possibilities to study effects of chemicals during embryo development with minimal confounding factors from the mother. In this study we explored blood plasma and allantoic fluid from chicken embryos as matrices for revealing metabolic changes caused by exposure to chemicals during embryonic development. Embryos were exposed via egg injection on day 7 to the environmental pollutant perfluorooctanoic acid (PFOA), and effects on the metabolic profile on day 12 were compared with those caused by GW7647 and rosiglitazone, which are selective agonists to peroxisome-proliferator activated receptor α (PPARα) and PPARγ, respectively. Analysis of the metabolite concentrations from allantoic fluid by Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) showed clear separation between the embryos exposed to GW7647, rosiglitazone, and vehicle control, respectively. In blood plasma only GW7647 caused a significant effect on the metabolic profile. PFOA induced embryo mortality and increased relative liver weight at the highest dose. Sublethal doses of PFOA did not significantly affect the metabolic profile in either matrix, although single metabolites appeared to be altered. Neonatal mortality by PFOA in the mouse has been suggested to be mediated via activation of PPARα. However, we found no similarity in the metabolite profile of chicken embryos exposed to PFOA with those of embryos exposed to PPAR agonists. This indicates that PFOA does not activate PPAR pathways in our model at concentrations in eggs and embryos well above those found in wild birds. The present study suggests that allantoic fluid and plasma from chicken embryos are useful and complementary matrices for exploring effects on the metabolic profile resulting from chemical exposure during embryonic development.

Oral health of individuals aged 3-80 years in Jönköping, Sweden during 40 years (1973-2013). II. Review of clinical and radiographic findings.

The aim of this epidemiological study performed in 2013 was to analyze various clinical and radiographic data on oral health and compare the results to those of four cross-sectional studies carried out 1973-2003. In 1973, 1983, 1993, 2003, and 2013 random samples of 1,000; 1,104; 1,078; 987; and 1,010 individuals, respectively, were studied. The individuals were evenly distributed in the age groups 3, 5, 10, 15, 20, 30, 40, 50, 60, 70, and 80o years. Eighty-year-olds were not included in 1973. All subjects were inhabitants of the city of Jönköping, Sweden. The clinical and radiographic examination assessed edentulousness, removable dentures, implants, number of teeth, caries, restorations, oral hygiene, calculus, periodontal status, and endodontic treatment. The frequency of edentulous individuals aged 40-70 years was 16, 12, 8, 1, and 0.3% in 1973, 1983, 1993, 2003, and 2013, respectively. No complete denture weareryounger than 80-years old was found in 2013. During the 40-year period, the mean number of teeth in the age groups 30-80 years increased. In 2013, the 60-year-olds had nearly complete dentitions. Implants were found in all age groups from 30 years of age. The total number of individuals with implants was 36 in 2013. This was higher than earlier surveys, 4 in 1993, and 18 in 2003. The percentage of children and adults without caries and restorations increased during the 40-year period. It was found that the percentage of caries-free 3- and 5-year-olds were 79% and 69%, respectively, of the individuals in 2013. In the age groups 10-20 years, the percentage of caries-free individuals increased between 2003 and 2013. In 2013, 43% of the 15-year-olds were completely free from caries and restorations compared to 20% in 2003. In all age groups 5-60 years, DFS was lower in 2013 compared to the earlier examinations.There was no major change in DFS between 2003 and 2013 in the age groups 70 and 80 years. The most obvious change was the decrease in number of FS over the 40-year period of time. Regarding crowned teeth the most clear changes between 1973 to 2013 were the decrease in percentage of crowned teeth in the age groups 40 and 50-year-olds. The percentage of endodontically treated teeth decreased between 1973 and 2013 in all age groups. In age groups 10-30-year-olds a major reduction from about 30% to 15% in mean plaque score was seen between 1973-2003. Only a minor change in plaque score was seen during the last decade. For the age groups 40 years and older, a decrease in the percentage of surfaces with plaque was observed between 2003-2013. The percentage of tooth sites with gingivitis was for 20 years and older about 40% in 1973. In 2013, the percentage was about 15%. The frequency of sites with gingivitis was generally lower in 2013 compared with the otheryears,1973-1993. The percentage of individuals with probing pocket depths > 4mm increased with age. Between 2003-2013 a clear reduction was seen in all age groups in frequency of individuals with probing pocket depth > 4mm. Over the 40-year period an increase in the number of individuals with no marginal bone loss and a decrease in the number of subjects with moderate alveolar bone loss were seen. The continuous improvement in oral health and the reduced need of restorative treatment will seriously affect the provision of dental helath care and dental delivery system in the near future.

Oral health of individuals aged 3-80 years in Jönköping, Sweden, during 40 years (1973-2013). I. Review of findings on oral care habits and knowledge of oral health.

The aim of the this study was to present data on oral care habits and knowledge of oral health in 2013, and to compare these data with results from a series of four previous cross-sectional epidemiological studies. All these studies were carried out in the city of Jönköping, Sweden, in 1973, 1983, 1993, 2003, and 2013. The 1973 study constituted a random sample of 1,ooo individuals evenly distributed in the age groups 3, 5, 10, 15, 20, 30, 40, 50, 60, and 70 years. The same age groups with addition of a group of 80-year-olds were included in the 1983, 1993, 2003, and 2013 studies, which comprised 1,104; 1,078; 987; and 1,010 individuals, respectively. A questionnaire about dental care habits and knowledge of oral health was used. The questionnaire contained the same questions in all the five studies, although some had to be slightly modernised during the 40-year period. During the period 1973-2013, a continous increase of individuals in the age group 20-60 years were treated by the Public Dental Service amounting to about 50%. Almost 70% of the 70- and 80-year-olds were treated by private practitioners. In 2013, 10-20% of the individuals in the age groups 30-40 years did not regularly visit neither Public Dental Service nor a private practitioner. The corresponding figures for the individuals 50-80 years old were 4-7%. Similar number of avoidance was reported in the previous studies. In the survey 2013, about 20-30% of the individuals in the age groups 20-50 felt frightened, sick, or ill at ease at the prospect of an appointment with the dentist. These findings were in agreement with the results from the surveys 1973-2003. Among the younger age groups, 0-15 years, a reduction in self-reported "ill at ease" was found in the surveys 2003 and 2013 compared to the previous surveys in this series. In 2013, the knowledge of the etiology of caries was known by about 60% of the individuals which was similar to that reported 1973-2003. Twenty per cent of the individuals stated that they did not know which etiological factors that causes caries. This percentage was equivalent during the period 1973-2013.About 85% of the individuals in all age groups brushed their teeth with fluoride tooth paste at least two times a day. These frequencies have gradually increased during the 40-year period. Around 40% in the age groups 50-80 years used toothpicks regularly in 2013. This is a about 1/3-1/2 less compared to 2003. In the age groups 20-40 years 3-14% used toothpicks for proximal cleaning in 2013. In 2013, about 35% of the individuals never consumed soft drinks, in comparison with 20% in 2003. In the age groups 3-20 years about 20% were consuming soft drinks every day or several times a week,which is a reduction by half compared to 2013.

Nine surface plasmon resonance assays for specific protein quantitation during cell culture and process development.

Quantitation of protein is essential during pharmaceutical development, and a variety of methods and technologies for determination of total and specific protein concentration are available. Here we describe the development of a streamlined assay platform for specific quantitation assays using surface plasmon resonance (SPR) technology. A total of nine different assays were developed using similar conditions, of which eight assays were for quantitation of different human blood plasma proteins (IgG, IgG1-4 subclasses, IgA, transferrin, and albumin) from a chromatography-based IgG plasma process. Lastly, an assay for monitoring the concentration of a recombinant monoclonal antibody during 13 days of CHO cell culturing was developed. Assay performances were compared with enzyme-linked immunosorbent assay (ELISA), nephelometry, ARCHITECT, and Cobas c501. SPR assays were shown to have higher sensitivity than analysis using nephelometry, ARCHITECT, and Cobas and to have significantly lower analysis and hands-on time compared with ELISA. Furthermore, the SPR assays were robust enough to be used for up to 12 days, allowing specific protein concentration measurement of a sample to be completed at line within 10 min. Using the same platform with only few varied parameters between different assays has saved time in the lab as well as for evaluation and presentation of results.

Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process.

BACKGROUND: Host cell proteins (HCP) should be carefully monitored in vaccine production. To achieve a reliable HCP estimation, a mixture of polyclonal antibodies (pAbs) with broad affinity would be of preference. Sensitive evaluations of the pAbs are therefore of value. METHODS: Column purification of specific HCPs with affinity to the anti-HCP pAbs was compared with Western blotting of the anti-HCP pAbs binding to filter bound total lysate. The anti-HCP pAbs were used in an HCP quantification analysis using surface plasmon resonance (SPR). Host cell derived impurities from an influenza vaccine process were analyzed using 2-D DIGE analysis. RESULTS: The Western blotting showed a similar HCP binding pattern of anti-HCP pAbs from immunizations using two adjuvants: CFA/IFA and AbISCO(®). From the column purification of HCPs, total proteins detectable were similar for all anti-HCP pAbs; however the immune response pattern differed significantly for the anti-HCP pAbs from the AbISCO(®) immunization. In the SPR HCP quantification assay the standard curve ranged from 0.3 to 40 µg/ml. The advantage of SPR compared with ELISA was the decreased hands on time and that the sample number was not limiting. The 2-D DIGE showed that most of the HCPs were removed at the clarification and virus capture step. DISCUSSION: Column purification of HCPs with affinity to the anti-HCP pAbs increased the sensitivity of affinity analysis compared with Western blotting and opened the possibility of further analysis. The anti-HCP pAbs did not interact with proteins in the virus; simplifying analysis of process samples using SPR. 2-D DIGE analysis gave a direct study of the impurity profile with the advantage of independence from antibody performance.

Hippocampal morphology in a rat model of depression: the effects of physical activity.

Accumulating in vivo and ex vivo evidences show that humans suffering from depression have decreased hippocampal volume and altered spine density. Moreover, physical activity has an antidepressant effect in humans and in animal models, but to what extent physical activity can affect hippocampal volume and spine numbers in a model for depression is not known. In this study we analyzed whether physical activity affects hippocampal volume and spine density by analyzing a rodent genetic model of depression, Flinders Sensitive Line Rats (FSL), with Magnetic Resonance Imaging (MRI) and ex vivo Golgi staining. We found that physical activity in the form of voluntary wheel running during 5 weeks increased hippocampal volume. Moreover, runners also had larger numbers of thin spines in the dentate gyrus. Our findings support that voluntary wheel running, which is antidepressive in FSL rats, is associated with increased hippocampal volume and spine numbers.

Albendazole causes stage-dependent developmental toxicity and is deactivated by a mammalian metabolization system in a modified zebrafish embryotoxicity test.

The zebrafish embryotoxicity test has previously been combined with an external metabolic activation system (MAS) to assess developmental toxicity of metabolites produced by maternal metabolism. Due to toxicity of MAS the exposure was limited to one early and short period. We have modified the method and included additional testing time points with extended exposure durations. Using the anthelmintic drug albendazole as a model substance, we demonstrated stage-dependent toxic effects at three windows of zebrafish embryo development, i.e. 2-3, 12-14 and 24-28h post fertilization, and showed that MAS, by metabolic deactivation, reduced the toxicity of albendazole at all time points. Chemical analysis confirmed that albendazole was efficiently metabolized by MAS to the corresponding sulfoxide and sulfone, which are non-toxic to zebrafish embryos. To conclude, the modified zebrafish embryotoxicity test with MAS can be expanded for assessment of metabolites at different developmental stages.

Activation of estrogen receptor alpha disrupts differentiation of the reproductive organs in chicken embryos.

Gonadal estrogen plays an important role in the differentiation of a female phenotype in birds. Exogenous compounds that interfere with estrogen signaling, for instance by binding to the estrogen receptors alpha and beta (ERα and ERß), are therefore potential disruptors of sexual differentiation in birds. The ERα agonist propyl-pyrazole-triol (PPT), the ERα antagonist methyl piperidino pyrazole (MPP) and the ERß agonist diarylproprionitrile (DPN) were used in the present study to explore the roles of the ERs in normal and disrupted sex differentiation in the chicken embryo. Activation of ERα by PPT caused disturbed differentiation of the reproductive organs in both sexes. In male embryos, PPT caused left-side ovotestis formation and retention of the Müllerian ducts. In female embryos, PPT caused retention of the right Müllerian duct (which normally regresses) and malformation of both Müllerian ducts. PPT also induced hepatic expression of mRNA for the estrogen-regulated egg yolk protein apoVLDL II. Notably, none of these effects were observed following treatment with DPN. ERα-inactivation by MPP counteracted the action of PPT but had little effect by its own. Our results indicate that ERα plays an important role in sex differentiation of the reproductive tract in female chicken embryos and show that ERα can mediate xenoestrogen-induced disturbances of sex differentiation.