Search

Phenibut (β − phenyl − γ−aminobutyric acid) is licensed for use as a medicine in countries outside of the European Union (EU), but has also been sold as a “food supplement” from online shops to the general public in the EU. We present a case of phenibut use in a 25-year-old female undergoing alcohol and drug addiction treatment. She reported using phenibut, which she had purchased readily over the internet as a “food supplement.” Our clinical laboratory located in a hospital in the same region received urine samples for analysis which confirmed ingestion of phenibut. Identifying and responding to new psychoactive substances (NPS) emerging on the drug markets poses a challenge to clinical and forensic drug testing. A comprehensive laboratory analysis approach can identify the use of multiple NPS, including those used as medicines, offering beneficial opportunities for drug treatment services and clinical laboratories to work together.

In the search for improved laboratory methods for the diagnosis of ethylene glycol poisoning, the in vivo formation of a glucuronide metabolite of ethylene glycol was hypothesized. Chemically pure standards of the β‐O‐glucuronide of ethylene glycol (EG‐GLUC) and a deuterated analog (d4‐EG‐GLUC) were synthesized. A high‐performance liquid chromatography and tandem mass spectrometry method for determination of EG‐GLUC in serum after ultrafiltration was validated. Inter‐assay precision (%RSD) was 3.9% to 15.1% and inter‐assay %bias was −2.8% to 12.2%. The measuring range was 2–100 μmol/L (0.48–24 mg/L). Specificity testing showed no endogenous amounts in routine clinical samples (n = 40). The method was used to analyze authentic, clinical serum samples (n = 31) from patients intoxicated with ethylene glycol. EG‐GLUC was quantified in 15 of these samples, with a mean concentration of 6.5 μmol/L (1.6 mg/L), ranging from 2.3 to 15.6 μmol/L (0.55 to 3.7 mg/L). In five samples, EG‐GLUC was detected below the limit of quantification (2 μmol/L) and it was below the limit of detection in 11 samples (1 μmol/L). Compared to the millimolar concentrations of ethylene glycol present in blood after intoxications and potentially available for conjugation, the concentrations of EG‐GLUC found in clinical serum samples are very low, but comparable to concentrations of ethyl glucuronide after medium dose ethanol intake. In theory, EG‐GLUC has a potential value as a biomarker for ethylene glycol intake, but the pharmacokinetic properties, in vivo/vitro stability and the biosynthetic pathways of EG‐GLUC must be further studied in a larger number of patients and other biological matrices.

The increase in opioid prescribing in many European countries over the last decade has raised concerns about associated diversion, overdose and mortality. Fentanyl is one of these synthetic opioids that is typically prescribed as a transdermal patch for pain that requires continuous pain relief and has been the focus of investigations due to reports of overdose and death.We report a case series of 14 drug addiction treatment entrants who entered treatment in one service located in the region of Southern Denmark from August 2015 to December 2015 for smoking fentanyl patches. Clients presented with difficulties breathing and pains in the lungs. The clients had a history of past opioid use, including heroin. Relapses resulted in treatment disengagement.Immunoassays for fentanyl were used in the service. In some cases, false negative results occurred. Clients’ urine samples were subsequently analysed in a collaborating laboratory. Seven clients tested positive for fentanyl. One client was positive for both fentanyl and heroin. Analyses were also positive for other opioids and metabolites in six clients, predominantly codeine and oxycodone. Results from confirmatory analysis contributed to clearer insights into clients’ drug histories, which facilitated personalised care plans consisting of opioid agonist therapy informed by confirmed drug use.In Denmark, prescription levels of fentanyl are high, which has been accompanied by observations of diversion and smoking in a smaller population. In addition to revision of inappropriate prescribing to reduce diversion, we recommend increased reliance upon confirmatory drug analysis in the addiction treatment sector in Denmark.

The emergence of an increasing number of new psychoactive substances (NPS) on the drug market requires a paradigm shift in drug testing. Immunoassay screening needs to be replaced with highly specific and sensitive analytical methods based on chromatography and mass spectrometry to produce accurate results, promote health and patient safety and collect data on the prevalence of NPS use, impact on public health and clinical aspects of NPS in Denmark. Development and implementation of new analytical methods currently present a major challenge for both clinical and forensic laboratories.

In Denmark, biennial population screening for colorectal cancer was introduced in 2014 for all aged 50–74 years. Five laboratories representative for the regional division of Denmark perform the immunochemical testing of faecal occult blood in the screening samples (iFOBT, OC-Sensor (Eiken Chemical, cut-off 100 µg/L)). In July 2016, a new agreement on the public post-delivery entailed an increased lag time (five days) from the screening participant drops the screening sample into a mail-box until sample arrival at the laboratories. Previous work had reported that a lag time above five days led to more false negative iFOBT tests. We investigated if this was true also under Danish conditions. We performed two stability tests; one with sample storage at 30 °C for 14 days (N = 60), and another with sample storage at room temperature for 13 days (N = 10). We extracted data from our laboratory information system (LABKA) on all iFOBT tests performed in the entire Central Denmark Region (N = 104,328 patients) during the last six months for each calendar year 2014–16. For each year, we computed the distribution of iFOBT tests below and above cut-off. Our stability tests showed no positive samples switching to false negative after storage; however, some negative samples turned false positive, especially at 30 °C. The data showed no change in the distribution of iFOBT tests below and above cut-off after July 2016. We found no evidence that an enhanced lag time increased the number of false negative iFOBT tests in the Danish screening program for colorectal cancer.

Aims To assess the incremental value of biomarkers, including N-terminal prohormone of brain natriuretic peptide (NT-proBNP), high-sensitivity troponin T (hs-TnT), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), growth differentiation factor 15 (GDF-15), and procollagen type 1 N-terminal propeptide (P1NP), in predicting incident cardiovascular events and mortality among asymptomatic individuals from the general population, beyond traditional risk factors, including fasting glucose and renal function (cystatin C), medication use, and echocardiographic measures. Methods and results Prospective population-based cohort study of 1324 subjects without a previous cardiovascular event, who underwent baseline echocardiography and biomarker assessment between 2002 and 2006. The clinical endpoint was the composite of myocardial infarction, invasively treated stable/unstable ischemic heart disease, heart failure, stroke, or all-cause mortality. Predictive capabilities were evaluated using Cox proportional-hazards regression, Harrell's concordance index (C-index), and net reclassification improvement. Median age was 66 (interquartile range: 60-70) years, and 413 (31%) were female. During median 8.6 (interquartile range: 8.1-9.2) follow-up years, 368 (28%) composite events occurred. NT-proBNP, hs-TnT, GDF-15, and IL-6 were significantly associated with outcome, independently of traditional risk factors, medications, and echocardiography ( p

Introduction: Misuse of opioid analgesics, in combination with diversion, dependence, and fatal overdoses, presents a serious problem for public health, which affects many countries worldwide. Within this context, tampering with opioids has been associated with serious harm. The aim of the present study was to assess the tampering potential of codeine combination analgesics on the market (containing codeine/non-opioid analgesics) by the extraction of codeine. Methods: Codeine was extracted from three combination formulations sold lawfully from licensed pharmacies without a medical prescription in Denmark and the UK. Extraction of codeine followed tampering procedures available on the Internet. The amounts of codeine and accompanying non-opioid analgesics in tampering products were analysed with liquid chromatography and tandem mass spectrometry (LC–MS/MS). Results: LC–MS/MS showed recoveries of the total amounts of codeine in tampering products of 81–84% from Product 1 (codeine/acetylsalicylic acid); 61–67% from Product 2 (codeine/ibuprofen); and 42–71% from Product 3 (codeine/paracetamol). Recoveries of non-opioid analgesics ranged between: 57–73% acetylsalicylic acid; 5.5–8.5% ibuprofen, and 5.0–9.2% paracetamol. Conclusion: With the tampering procedures used, high amounts of codeine were separated from the accompanying analgesics in some, but not in all of the codeine containing formulations. Evidence-based medicine regulation, treatment for opioid dependence, and information to minimise risks to the public are essential components of an effective public health strategy to address the harms of tampering and misuse. Funding: Marie Pedersen and Jensine Heiberg Foundation.

Tampering with opioid analgesics for misuse is an emerging and complex drug problem, responsible for serious harms including dependence and overdose. There is a need to better detect and monitor signals of misuse in order to produce better data to inform public health.

By electro-immunoprecipitation methods it was shown that antigen Tp-4 of Treponema pallidum, antigen TR-c of the Reiter treponeme and 'Common Antigen' of Pseudomonas aeruginosa are immunologically cross-reactive. This finding establishes a taxonomic relationship between T. pallidum and a wide range of bacteria. The cross-reactivity explains some false positive reactions in serodiagnostic treponemal tests for syphilis. The cross-reactivity may also play a role in a possible 'normal' immunity against infection with T. pallidum induced by antigens by the normal human bacterial flora.

CONTEXT: Thyroid hormones are important developmental factors and levels should be adequate both in the pregnant woman and in the fetus. However, there is no consensus on maternal thyroid test reference limits in early pregnancy.OBJECTIVE: Estimation of week-to-week changes in and predictors of TSH and free T4 (fT4) reference limits in the first trimester of pregnancy.DESIGN: Measurement of TSH and fT4 in biobank sera collected in pregnancy weeks 5-19 from a random sample of the Danish National Birth Cohort that enrolled 101 032 pregnant in 1996-2002.SETTING: National cohort of pregnant women.PARTICIPANTS: Healthy participants (n = 6671) were identified and individual characteristics retrieved using interview data and data from Danish national health registers.INTERVENTION(S): None.MAIN OUTCOME MEASURE(S): Reference limits for TSH and fT4 in each first trimester pregnancy week and predictors of these reference limits.RESULTS: TSH reference limits were very variable. Up to and including week 6, nonpregnancy reference limits could be used. In weeks 9-12, TSH upper reference limit was approximately 0.4 mU/L lower than the nonpregnancy upper limit. The TSH lower reference limit was approximately 0.1 mU/L. fT4 variations were reverse to those of TSH, but changes were small with approximately 4% higher reference limits during the weeks 9-12. TSH upper reference limit was lower in multiparous women and women with lower iodine intake but higher in obese women. fT4 was lower in smokers.CONCLUSIONS: TSH reference limits differ widely in the first trimester of pregnancy. The use of a uniform set of reference limits is an inordinate simplification that will lead to frequent misclassification and possibly to incorrect choice of therapy.

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted (entrained) by light via the retinohypothalamic tract (RHT). The RHT contains two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which are believed to mediate the phase-shifting effects of light on the clock. In the present study we have elucidated the role of PACAP in light-induced phase shifting at early night in hamsters and shown that (i) light-induced phase delay of running-wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6-38) or by immunoblockade with a specific anti-PACAP antibody injected intracerebroventricularly before light stimulation; (ii) PACAP administered close to the SCN was able to phase-delay the circadian rhythm of running-wheel activity in a similar way to light; (iii) PACAP was present in the hamster RHT, colocalized with melanopsin, a recently identified opsin which has been suggested to be a circadian photopigment. The findings indicate that PACAP is a neurotransmitter of the RHT mediating photic information to the clock, possibly via melanopsin located exclusively on the PACAP-expressing cells of the RHT.

New methods were developed and validated to determine the identity, contents, and purity of samples of melanotan II, a synthetic melanocortin receptor agonist, sold in vials as injectable skin-tanning products that were purchased from three online shops. Methods were based on liquid chromatography with ultra-violet detection (LC-UV) at wavelength 218 nm, and tandem mass spectrometric detection (MS/MS) after collision-induced fragmentation of the double charged [M+2H](2+) precursor ion (m/z 513). Identification of melanotan II was verified by correct chromatographic retention time, and relative abundance ratios of five qualifying fragment ions. LC-UV was used to quantify melanotan II as well as impurities. Method validation was performed with reference to guidelines for assessing active substances in authorized medicinal products to reach acceptable accuracy and precision. Vials from two shops contained unknown impurities ranging from 4.1 to 5.9%; impurities from one shop were below the quantification limit. The total amount of melanotan II in vials ranged between 4.32 and 8.84 mg, although each shop claimed that vials contained 10 mg melanotan II. A broad range of drugs used for enhancement purposes can be obtained from the illicit market. However, users of these drugs may be exposed to a range of potential harms, as shown in this study, given that these products are manufactured, distributed and supplied from an illicit market. Copyright © 2014 John Wiley & Sons, Ltd.

AIMS: To examine whether increasing fasting plasma glucose (FPG) levels were associated with worsening left ventricular (LV) diastolic function, independently of LV mass index (LVMI) in elderly, otherwise healthy subjects.METHODS AND RESULTS: We tested cross-sectional associations between echocardiographically determined averaged E/é ratio/diastolic function, LVMI, cardiovascular risk factors, and FPG categorized as normal (NFG), impaired (IFG), and new-onset diabetes mellitus (DM), in 483 men and 208 women aged 56-79years without overt cardiovascular disease, who received no cardiovascular, anti-diabetic, or lipid-lowering drugs and had a preserved LV ejection fraction >50%. Median E/é was significantly higher among subjects with diabetes than those without (8 vs. 7; p=0.03), as was the prevalence of grade 2 or 3 diastolic dysfunction (25% vs. 16%; p=0.02). E/é and diastolic function were significantly associated with LVMI (p≤0.002), but not FPG category, on multivariable analysis. However, interaction analyses revealed that increasing LVMI was primarily associated with worsening diastolic function (higher E/é) in subjects with FPG>6mmol/L (β=0.005 for IFG and DM vs. 0.001 for NFG; p=0.02), whereas increasing systolic blood pressure was primarily associated with worsening diastolic function (higher E/é) in subjects with FPG≤6.9mmol/L (β=0.005 for NFG and 0.003 for IFG vs. -0.001 for DM; p=0.001).CONCLUSION: Diastolic dysfunction was significantly more prevalent among patients with DM than those without. The importance of LVMI increased, but the importance of systolic blood pressure decreased with higher FPG category.

As Ammatuna and Nijziel note,1 obtaining an accurate and comprehensive drug history is crucial in the treatment of patients presenting with symptoms that can potentially be attributed to the use of anabolic steroids, including renal disorders. Most people who use anabolic steroids obtain these drugs from the illicit market.2 Here, many products are manufactured in ‘underground laboratories’ operating outside the formal parameters of the production of licensed medicinal products.3 In the UK, investigations by the Medicines and Healthcare …

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis , and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae . Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis , was sequenced from 18 rifampin-resistant Legionella isolates representing four species ( L. bozemanii , L. longbeachae , L. micdadei , and L. pneumophila ), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae .

To study the structure and function of the Legionella flagellum, we screened a genomic L. micdadei library in Escherichia coli for expression of the flagellin (Fla) subunit. One recombinant clone, JM105 (pHI5588), producing a truncated Fla protein of 40.5 kDa was identified. The plasmid pHI5588 carried a L. micdadei DNA insert of 5 kb, containing ca 95% of the fla gene. The complete DNA sequence of the L. micdadei fla gene was obtained by combining sequence data from pHI5588 with results using a polymerase chain reaction-based system for genome walking (vectorette PCR). The L. micdadei fla gene shared a high degree of homology with other flagellin genes in the amino- and carboxy termini, whereas the central region was found to be nonconserved. The fla sequence will facilitate the cloning of Fla proteins from other Legionella species and the study of flagella in the pathogenesis of Legionnaires' disease.

Background This study compared NT-proBNP levels and the association with cardiovascular markers between Africans and Caucasians from South Africa. Methods This cross-sectional study involved 201 Africans and 255 Caucasians from the North West province, South Africa. Serum NT-proBNP concentrations, blood pressure, pulse wave velocity and arterial compliance were measured. Results NT-proBNP levels were significantly higher (P < 0.001) in Africans than Caucasians, also after adjusting for gender, body mass index (BMI) and pulse wave velocity (P = 0.008). This significant difference became borderline significant after adjusting for systolic blood pressure (SBP) (P = 0.060), and non-significant after adjusting for arterial compliance (P = 0.35). In single regression, a significant positive correlation of NT-proBNP with SBP (r = 0.26; P < 0.001) and pulse pressure (PP) (r = 0.28; P < 0.001) were shown for Africans only. After multiple adjustments, the associations of NT-proBNP with SBP and PP remained significant in Africans (SBP: β = 0.187, P < 0.01; PP: β = 0.234, P < 0.001), with no significant associations in Caucasians. Conclusions NT-proBNP levels were higher in Africans than Caucasians, independently of BMI and gender. This difference was partly driven by higher SBP and lower arterial compliance in Africans. NT-proBNP was persistently associated with SBP and PP in Africans, but not in Caucasians. These associations may suggest early vascular changes contributing to cardiac alterations in Africans. http://dx.doi.org/10.1016/j.hlc.2011.10.009

Analytical methods for pharmacokinetic studies with quantification of the highly potent anaesthetic drug fentanyl in serum must be rugged, sensitive, precise and accurate. To address these analytical demands and facilitate an automated approach to sample clean-up, a new method was developed for human serum using back-flush columnswitching and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). Prior to injection, protein precipitation in trichloroacetic acid was performed in each sample. Samples were diluted with internal standard (D5-fentanyl) in 10% (w/v) trichloroacetic acid, kept at 4oC for 20 min and centrifuged followed by injection of 40μL supernatant onto a BioTrap 500 MS extraction column. Using a time programmed six-port valve switch, the extracted drug was back-flushed onto a Zorbax SB-Aq analytical column, gradient eluted and finally detected after electrospray ionisation with multiple reaction monitoring (MRM) of the transitions m/z 337 → m/z 188 and m/z 342 → m/z 188 for fentanyl and D5-fentanyl, respectively. Mean inter-assay accuracy (n=5) ranged from 93 to 101 % and inter-assay precision (n=5) ranged from 3.7 to 9.4% (C.V.). The method was used in a preliminary study of 4 patients after application of transdermal patches and found fully applicable for future pharmacokinetic studies. This is the first on-line, column-switching LC-MS/MS method validated and demonstrated suitable for quantification of fentanyl in human serum.

A 2.7 kb DNA fragment encoding the 60 kDa common antigen (CA) and a 13 kDa protein of Legionella micdadei was sequenced. Two open reading frames of 57677 and 10456 Da were identified, corresponding to the heat shock proteins GroEL and GroES, respectively. Typical −35, −10, and Shine-Dalgarno heat shock expression signals were identified upstream of the L. micdadei groEL gene. Further upstream, a poly-T region, also a feature of the σ32-regulated Escherichia coli groELS heat shock operon, was found. Despite the high degree of homology of the expression signals in E. coli and L. micdadei , Western blot analysis with an L. micdadei specific anti-groEL antibody did not reveal a significant increase in the amount of the GroEL protein during heat shock in L. micdadei or in the recombinant E. coli expressing L. micdadei GroEL.

Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus, and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. In the mammalian gonads, clock genes have been reported in the testes, but the expression pattern is developmental rather than circadian. Here we investigated the daily expression of the two core clock genes, Per1 and Per2, in the rat ovary using real-time RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Both Per1 and Per2 mRNA displayed a statistically significant rhythmic oscillation in the ovary with a period of 24 h in: 1) a group of rats during proestrus and estrus under 12-h light,12-h dark cycles; 2) a second group of rats representing a mixture of all 4 d of the estrous cycle under 12-h light,12-h dark conditions; and 3) a third group of rats representing a mixture of all 4 d of estrous cycle during continuous darkness. Per1 mRNA was low at Zeitgeber time 0–2 and peaked at Zeitgeber time 12–14, whereas Per2 mRNA was delayed by approximately 4 h relative to Per1. By in situ hybridization histochemistry, Per mRNAs were localized to steroidogenic cells in preantral, antral, and preovulatory follicles; corpora lutea; and interstitial glandular tissue. With newly developed antisera, we substantiated the expression of Per1 and Per2 in these cells by single/double immunohistochemistry. Furthermore, we visualized the temporal intracellular movements of PER1 and PER2 proteins. These findings suggest the existence of an ovarian circadian clock, which may play a role both locally and in the hypothalamo-pituitary-ovarian axis.

Circadian rhythms are daily adjusted to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Recent studies indicate that melanopsin, a newly identified opsin-like molecule, is involved in the light responsiveness of retinal ganglion cells (RGCs) constituting the RHT. In the present study, we examined the expression of melanopsin at the mRNA and protein level during a day/night cycle and during prolonged periods of light and darkness in the retina of albino Wistar rats. We observed a diurnal change in melanopsin, with mRNA level being highest at early subjective night and protein level highest at late subjective day. Prolonged exposure to darkness significantly increased melanopsin mRNA level as early as the first day, and the expression continued to increase during 5 d in darkness. The decrease in mRNA level during exposure to constant light was slower. After 48 h of light, the melanopsin mRNA level was significantly reduced, and an almost undetectable level was found after 5 d. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 d. By use of immunohistochemistry, we showed that darkness increased the amount of protein in the dendritic processes, resulting in a dense network covering the entire retina. Constant light decreased melanopsin immunostaining time dependently, beginning in the distal dendrites and progressing to the proximal dendrites and the soma. Our observations suggest that the intrinsic light-responsive RGCs adapt their expression of the putative circadian photopigment melanopsin to environmental light and darkness.

PURPOSE. The putative circadian photoreceptor melanopsin is found in rodents in a subpopulation of intrinsic light-sensitive retinal ganglion cells (RGCs) constituting the retinohypothalamic tract (RHT). The study was conducted to determine whether melanopsin is expressed in the human retina and costored with the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP), a marker for the RHT, projecting to the suprachiasmatic nucleus (SCN). Furthermore, whether melanopsin expression is conserved in retinas of blind patients with severe retinal degeneration was investigated. METHODS. In situ hybridization and immunohistochemistry was used to demonstrate melanopsin synthesis in human eyes of 17 donors and two postmortem hypothalami containing the SCN. The coexistence of melanopsin and PACAP in elements of the retinohypothalamic tract was studied by dual-labeling immunocytochemistry. RESULTS. Melanopsin expression was found in a subpopulation of RGCs located in the ganglion cell layer and displaced in the inner nuclear cell layer. Melanopsin-containing cells comprised approximately 0. 8% of all RGCs, with a distinct morphology characterized by two to four dendritic processes constituting a panretinal network. Melanopsin immunoreactivity was primary present at perikaryal boundaries and neuronal processes and to some extent also in the cytoplasm. PACAP and melanopsin were colocalized in the RGCs and PACAP-containing nerve fibers, seemingly innervating the retinorecipient part of the SCN. Melanopsin-expressing RGCs were conserved in retinas of blind patients with severe degeneration of the outer and/or inner layers. CONCLUSIONS. Given the expression of melanopsin in PACAPcontaining RGCs of the human RHT, this photoreceptor is a likely first base in the chain of events leading to photoentrainment of both normal and blind people. (Invest Ophthalmol Vis Sci. 2004;45:4202‐4209) DOI:10.1167/iovs.04-0313

Despite severe degeneration of its eyes, the blind subterranean mole rat, Spalax, is able to adjust circadian rhythms to the environmental light/dark cycle due to a conserved retinohypothalamic tract (RHT). The photopigment mediating the circadian photoreception and it cellular localisation is unknown in the Spalax retina. Here we show, using in situ hybridization and immunohistochemistry, that melanopsin, a recently identified opsin, is expressed in retinal ganglion cells which also co-store PACAP, a neurotransmitter of the RHT. The melanopsin-component of retinal ganglion cells in the Spalax retina is well conserved resulting in a relatively higher density of melanopsin positive cells per area compared to the rat. The results show that the Spalax, as sighted animals expresses melanopsin in ganglion cells projecting to the circadian clock supporting a role of melanopsin as a circadian photopigment.

Mammalian circadian rhythms generated in the hypothalamic suprachiasmatic nuclei are entrained to the environmental light/dark cycle via a monosynaptic pathway, the retinohypothalamic tract (RHT). We have shown previously that retinal ganglion cells containing pituitary adenylate cyclase-activating polypeptide (PACAP) constitute the RHT. Light activates the RHT via unknown photoreceptors different from the classical photoreceptors located in the outer retina. Two types of photopigments, melanopsin and the cryptochromes (CRY1 and CRY2), both of which are located in the inner retina, have been suggested as "circadian photopigments." In the present study, we cloned rat melanopsin photopigment cDNA and produced a specific melanopsin antibody. Using in situ hybridization histochemistry combined with immunohistochemistry, we demonstrate that the distribution of melanopsin was identical to that of the PACAP-containing retinal ganglion cells. Colocalization studies using the specific melanopsin antibody and/or cRNA probes in combination with PACAP immunostaining revealed that melanopsin was found exclusively in the PACAP-containing retinal ganglion cells located at the surface of somata and dendrites. These data, in conjunction with published action spectra analyses and work in retinally degenerated (rd/rd/cl) mutant mice, suggest that melanopsin is a circadian photopigment located in retinal ganglion cells projecting to the biological clock.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha. This vaccination approach leads to a T-cell-dependent polyclonal and sustainable anti-TNF-alpha autoantibody response that declines upon discontinuation of booster injections. The autoantibodies are elicited by injecting modified recombinant TNF-alpha molecules containing foreign immunodominant T-helper epitopes. In mice immunized with such molecules, the symptoms of experimental cachexia and type II collagen-induced arthritis are ameliorated. These results suggest that vaccination against TNF-alpha may be a useful approach for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.

Combining the electrophoretic principles of counter current immunoelectrophoresis and deflection of line precipitates in line immunoelectrophoresis provides a new technique for quantitative determination of antibodies against specific antigens (CCLD electrophoresis), even if no purified antigen or monospecific antibodies are available for construction of the detection system. We have used the method for quantitative determination of antibodies against the flagellum of Treponema phagedenis biotype Reiter and compared the diagnostic potential of this method in the diagnosis of syphilis with an ELISA method for the quantification of IgG antibodies against the flagellum. The CCLD electrophoresis could be optimized to a diagnostic performance very similar to that achieved using the ELISA method.

As the first step in a cooperative effort to standardize the identification of the polypeptides of Treponema pallidum subsp. pallidum, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 of the major polypeptide bands, the cross-identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two-dimensional electrophoresis provided an improved means of separating and characterizing T. pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two-dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two-dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T. pallidum proteins.

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae recombinants were frequently found expressing an immunodominant 65 kDa protein antigen. In this study polyclonal and monoclonal antibodies against the 65 kDa antigen were found to react with a variety of different bacteria. Furthermore it is shown that the 65 kDa mycobacterial protein belongs to the family of antigens previously designated ‘common antigen’ due to their presence in a large variety of bacterial species. The molecular weight of this common antigen in 17 bacterial species was determined and it varied from 59 to 65 kDa. These bacteria included Gram-negative, Gram-positive and archae-bacteria, indicating that this antigen consists of members of an evolutionary well-conserved protein family. The 65 kDa protein was located in the cytoplasmic fraction of both Escherichia coli K12 and M. bovis BCG. Its function for the bacterial cell is presently unknown. The immunological relatedness of this common antigen to the MbaA protein might indicate a role in the etiology of rheumatoid arthritis, as was recently suggested for the mycobacterial 65 kDa antigen.

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15–25 kilobase pairs (kb)) were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli -absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly with a monospecific rabbit antiserum raised against L. micdadei“common antigen” (CA), and an additional 13 K L. micdadei protein. The region encoding these two proteins from the 17 kb recombinant plasmid (pBA 2) was subcloned in pBGS 18+. The DNA sequence of the CA encoding region in the 2.7 kb subcloned fragment will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function.

The immunochemical properties of the Treponema pallidum antigen TpD, as expressed by Escherichia coli K12, was investigated by crossed immunoelectrophoresis in which an affinity-purified antibody to this antigen was used. Two immunologically cross-reacting components of TpD with different mobility were demonstrated. Affinity-purified antibodies were used in obtaining purified TpD and in determining the cellular localization of TpD in T. pallidum by immunoelectron microscopy. TpD was localized on the surface of methanol-fixed T. pallidum. Twenty sera from patients with secondary syphilis and 20 sera from nonsyphilitics were examined in crossed immunoelectrophoresis. All sera from patients with secondary syphilis and none from nonsyphilitics contained antibodies to the TpD components. Because TpD seems to be surface associated and a major immunogen during infection with T. pallidum, this antigen might be useful for development of a vaccine against syphilis and for development of improved methods for serodiagnosis of syphilis.

Variants of pregnancy specific beta 1-glycoprotein have been described previously. These variants seem to cause artificially low levels when measured by radioimmunoassay. We demonstrate that sera with indistinct precipitates in electroimmunoassay give less steep dose-response curves in radioimmunoassay than do sera with well defined precipitates. Until parallel dose-response curves are demonstrated for all variants, previously published data must be treated with reserve.

SUMMARY: A library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the ‘common antigen' (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.

Thirteen rabbits were immunized three times at weekly intervals with 50 micrograms of flagella purified from Treponema phagedenis biotype Reiter. Fourteen rabbits were inoculated in the same way with a placebo preparation. Rabbits immunized with the flagella developed an immune response to the flagella but showed no statistically significant prolongation of incubation time or diminution of lesion severity when challenged intradermally with 4 X 10(3) Treponema pallidum organisms.

Quantitative crossed immunoelectrophoresis was used to evaluate the antigenic similarity of Pseudomonas aeruginosa and Pseudomonas cepacia GroEL proteins. We found that the two proteins showed 75% identity. By using a panel of monoclonal antibodies against the P. aeruginosa GroEL protein, we identified 10 monoclonal antibodies which cross-reacted with the P. cepacia GroEL protein and 21 monoclonal antibodies which recognized type-specific epitopes on the P. aeruginosa GroEL protein. In crossed immunoelectrophoresis two different fractions of GroEL reactive material could be resolved. These fractions showed a reaction of partial identity. Examination of the two immunoprecipitates by Western blotting, showed that both fractions consisted of anti-60 kDa GroEL reactive protein. One fraction, in addition, contained LPS with a characteristic ‘ladder’ reaction in modified Western blotting. We therefore conclude that this fraction represents a complex between LPS and GroEL.

Background Social inequalities has been shown for participation in colorectal cancer screening and recently in the initial stool sample blood test. If these differences persist at follow-up colon examination after a positive stool test, it would suggest that social inequality in screening may be greater than the inequality observed in initial stool sample blood test. Methods All data were derived from national registers. Using logistic regression analyses, odds of non-participation for follow-up colon examination were estimated based on age group, educational level, income quartile, immigration status and marital status in men and in women, who had participated in initial stool sample test for blood with a positive result. Results Among 20 849 men and 16 565 women invited for follow-up colonoscopy in the period 2014–15, 10.63 and 11.37%, respectively, did not attend. In men, odds of non-participation were higher in the eldest, those with lower income and lower educational level, in immigrants and in singles. Odds ratio (OR) in males of highest income quartile was 0.54 [95% confidence interval (CI) 0.46; 0.63] compared with lowest income quartile. In women, the differences were not as large. OR in females of highest income quartile was 0.73 (95% CI 0.61; 0.87) compared with lowest income quartile. Conclusion Sociodemographic differences in odds of non-participation exist in follow-up colon examination in the Danish colorectal cancer screening. Differences were evident in all subgroups of the male population. The same patterns were seen in women. Social inequalities in participation for follow-up colon examination can increase overall social inequality and consequently, lead to health disparities.