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Objectives Patient-reported outcome measures (PROMs) are evaluated in randomized controlled trials (RCTs) in patients with systemic lupus erythematosus (SLE), but not widely used in clinical practice. However, interest in incorporating PROMs into the management of SLE is increasing as PROMs provide a unique insight into the patient’s perception of lupus disease activity. The objective was to assess agreement in PROMs answered using a web app versus an outpatient touchscreen among patients with SLE. Methods In a crossover RCT, SLE patients answered the following PROMs in a random order using the web app and the outpatient touchscreen: Systemic Lupus Erythematosus Activity Questionnaire (SLAQ) Global Health, SLAQ Symptom, SLAQ Total, SLAQ Worsening, Pain Visual Analog Scale (VAS), Fatigue VAS, Patient Global Health VAS, Health Assessment Questionnaire Disability Index (HAQ-DI), Patient Acceptable Symptom State (PASS), and an Anchoring Question. Equivalence between the two device types was demonstrated if the 95% confidence interval (95% CI) of the difference in PROM scores was within the prespecified equivalence margin. Agreement between the two device types was assessed using mixed linear models. Results Thirty-four patients with SLE were included. Equivalence was demonstrated between the two device types for SLAQ Global Health with a difference of −0.21 (95% CI: −0.65 to 0.23). Moreover, equivalence was also found for HAQ-DI, Pain VAS, and Fatigue VAS whereas only comparability within the limits of the Minimal Clinically Important Difference (MCID) was demonstrated for VAS Patient Global Health. Statistical comparability was demonstrated for SLAQ Total, SLAQ Worsening, PASS, and Anchoring Question (no predefined MCID/equivalence margins available). However, a statistically significant difference between device types was observed for the SLAQ Symptom of −0.56 (95% CI: −1.10 to −0.01). The difference was, however, very small when considering the scale range of 0–24; thus, it was not judged to be of clinical relevance. Preference for the web app was very high (91.2%). Conclusion For the first time ever, equivalence and comparability between two electronic device types for various PROMs were demonstrated among patients with SLE. Implementation of the device is expected to improve the management of SLE.

BackgroundSystemic lupus erythematosus (SLE) has been associated with more than 100 genetic loci. This parallels positively to the clinical diversity that is reflected by the classification of SLE.ObjectivesWe aimed to investigate associations between disease manifestations of SLE and risk gene variants relevant to Danish subjects of European ancestry.MethodsWe included 427 SLE patients of European ancestry similar to previous reports.[1] We also included 89,699 controls from the Danish Blood Donor Study Genomic Cohort. SLE risk loci in this population were identified by genome-wide association methodology and hereafter correlated to cumulative occurrence of SLE classification items.ResultsFourteen variants mapped to the following genes: NCF2, STAT4, TNPO3/TPI1P2, IRF5, and ITGAM, were significantly associated (pThe five lead variants were associated (pNCF2: proteinuria and anti-phospholipid antibodies, STAT4: arthritis, serositis, neurologic disorder, lymphopenia, and anti-Smith antibodies, IRF5: seizures and proteinuria, TNPO3: proteinuria, and ITGAM: photosensitivity (Table 2).ConclusionOur findings support the future use of select, relevant genetic markers in predicting various SLE phenotypes.References[1]Leffers HCB, Troldborg A, Voss A, et al. Smoking associates with distinct clinical phenotypes in patients with systemic lupus erythematosus: a nationwide Danish cross-sectional study. Lupus Sci Med 2021;8(1).Table 1.Associations between five SLE risk loci and specific disease manifestations in 427 Danish patients with SLE*.NCF2STAT4IRF5TNPO3ITGAMrs17849502_Trs7574865_Trs4728142_Ars13239597_Ars11860650_TN (%)Malar rash233 (55%)1.28 (0.84-1.96)0.83 (0.62-1.11)1.01 (0.74-1.38)1.44 (0.97-2.12)1.14 (0.80-1.61)Discoid rash46 (11%)1.49 (0.81-2.73)0.90 (0.56-1.45)1.01 (0.62-1.66)1.16 (0.63-2.12)0.76 (0.42-1.41)Photosensitivity219 (51%)0.96 (0.63-1.46)1.09 (0.81-1.47)0.98 (0.71-1.34)0.84 (0.57-1.25)0.67 (0.47-0.97)Oral ulcers132 (31%)0.96 (0.61-1.50)0.90 (0.65-1.23)0.83 (0.60-1.16)1.30 (0.87-1.96)1.43 (0.99-2.05)Non-erosive Arthritis342 (80%)0.84 (0.52-1.37)1.49 (1.02-2.18)0.93 (0.63-1.36)1.04 (0.64-1.68)1.16 (0.74-1.80)Serositis-Pleuritis124 (29%)0.63 (0.38-1.05)1.38 (1.01-1.89)1.22 (0.87-1.72)0.85 (0.56-1.29)0.84 (0.57-1.24)-Pericarditis72 (17%)0.75 (0.41-1.40)1.35 (0.93-1.96)1.05 (0.70-1.58)1.15 (0.70-1.89)1.09 (0.70-1.72)Persistent proteinuria158 (37%)1.63 (1.07-2.49)1.08 (0.80-1.46)0.68 (0.49-0.94)1.74 (1.16-2.61)1.09 (0.76-1.57)Neurologic disorder-Seizures23 (5%)1.58 (0.75-3.35)1.49 (0.80-2.76)2.10 (1.04-4.25)0.61 (0.26-1.44)0.93 (0.42-2.06)-Psychosis8 (2%)0.76 (0.097-5.87)2.77 (0.94-8.15)0.35 (0.10-1.23)0 (0)2.96 (0.85-10.3)Haematologic disorder-Haemolytic anaemia38 (9%)0.78 (0.34-1.76)1.37 (0.85-2.22)0.75 (0.44-1.29)1.11 (0.57-2.19)1.24 (0.70-2.20)-Leukopenia130 (30%)1.04 (0.67-1.61)1.19 (0.87-1.63)1.00 (0.72-1.39)0.90 (0.60-1.37)0.94 (0.64-1.37)-Lymphopenia228 (53%)0.95 (0.63-1.44)1.35 (1.01-1.81)0.95 (0.70-1.29)1.16 (0.79-1.70)1.09 (0.77-1.54)-Thrombocytopenia102 (24%)1.42 (0.91-2.22)0.84 (0.60-1.18)0.83 (0.58-1.18)1.35 (0.86-2.11)0.91 (0.60-1.37)Immunologic disorder-anti-DNA ab.330 (77%)0.69 (0.44-1.09)1.02 (0.72-1.44)0.94 (0.65-1.35)0.97 (0.62-1.53)1.08 (0.71-1.65)-anti-Smith ab.44 (10%)1.44 (0.79-2.64)1.58 (1.00-2.49)1.23 (0.73-2.07)1.47 (0.80-2.69)1.07 (0.61-1.84)-anti-phospholipid ab.183 (43%)1.63 (1.07-2.49)1.05 (0.79-1.41)0.84 (0.61-1.14)1.14 (0.77-1.68)1.14 (0.80-1.62)* Logistic regression models for each manifestation included all five lead variants (multivariate) and were adjusted for age and sexDisclosure of InterestsHenrik Leffers: None declared, David Westergaard: None declared, Saedis Saevarsdottir: None declared, Ingileif Jonsdottir: None declared, Ole Birger Pedersen: None declared, Anne Troldborg: None declared, Anne Voss: None declared, Salome Kristensen: None declared, Jesper Lindhardsen: None declared, Prabhat Kumar: None declared, Asta Linauskas: None declared, Lars Juul: None declared, Niels Steen Krogh: None declared, Bent Deleuran: None declared, Lene Dreyer Speakers bureau: Speakers bureau: Eli Lilly, Galderma and Janssen, Grant/research support from: from BMS outside the present work, Michael Schwinn: None declared, Lise wegner Thørner: None declared, Lotte Hindhede: None declared, Christian Erikstrup: None declared, Henrik Ullum: None declared, Søren Brunak Shareholder of: SB has ownerships in Intomics A/S, Hoba Therapeutics Aps, Novo Nordisk A/S, Lundbeck A/S, Kari Stefansson: None declared, Karina Banasik: None declared, Søren Jacobsen: None declared

BackgroundNephritis (LN) in systemic lupus erythematosus (SLE) is still a major determinant of poor prognosis[1].The vast majority of LN occurs in proximity to the SLE diagnosis[2]. Identification of individuals at high risk, especially early onset SLE, is therefore warranted. Inclusion of risk factors prior to the SLE diagnosis may thus be of importance to enable sufficient risk factor profiling. SLE-patients seem to cluster according to clinical and serological phenotypes suggesting distinct disease trajectories[3-5].ObjectivesTo determine if incident proteinuria associated with the debut age of non-renal SLE characteristics.MethodsData of SLE patients from six Danish centers were obtained from the Danbio-database from 2017 – 2020. The occurrence and timing of proteinuria was compared with first time onset of any non-renal manifestations as defined by the 1997 American College of Rheumatology Classification Criteria. Cox-regression models were used to identify risk factors for incident proteinuria. Time from first occurring non-renal manifestation to incident proteinuria or censoring defined time at risk. Covariates were eliminated if p >0.01 in a ‘backwards’ manner. After the model reduction process p-values Results586 SLE patients, mainly white (94%) women (88%), mean age at inclusion of 34.6 years (standard deviation, SD = 0.6 years) and observed for a mean of 14.9 years (SD =0.5 years), were recruited. The cumulative prevalence of proteinuria was 40%. Male gender hazard ratio, HR = 1.35 (range 0.77-2.35), p=0.009, lymphopenia HR = 1.77 (range 1.24-2.52), p=0.005 were associated with incident proteinuria. In contrast, patients with discoid rash had lower risk of incident proteinuria HR 0.42 (range 0.21-0.83), p=0.01. Male patients with lymphopenia had the highest risk of proteinuria with a one-, 5- and 10-year risk of proteinuria ranging from 9-27%, 34-75% and 51-89 %, depending on the age at presentation (debut at 20, 30, 40 or 50 years). The corresponding risk-profiles for women with lymphopenia were 3-9%, 8-34% and 12-58%, respectively, as illustrated in Figure 1.ConclusionThe occurrences of lymphopenia and discoid rash were oppositely associated with risk of incident proteinuria and the risk effects varied according to gender and patient age at onset of these manifestations. Thus, the risk of proteinuria may not be constant but could vary according to presentation of non-renal manifestations that may call for a differentiated clinical follow-up. Based on these findings, we suggest that the debut age of known prognostic factors, even prior to the SLE diagnosis should be considered when designing prognostic statistical models.References[1]Faurschou, M., et al., Prognostic factors in lupus nephritis: diagnostic and therapeutic delay increases the risk of terminal renal failure. J Rheumatol, 2006. 33(8): p. 1563-9.[2]Hanly, J.G., et al., The frequency and outcome of lupus nephritis: results from an international inception cohort study. Rheumatology (Oxford), 2016. 55(2): p. 252-62.[3]Diaz-Gallo, L.M., et al., Four Systemic Lupus Erythematosus Subgroups, Defined by Autoantibodies Status, Differ Regarding HLA-DRB1 Genotype Associations and Immunological and Clinical Manifestations. ACR Open Rheumatol, 2022. 4(1): p. 27-39.[4]Jacobsen, S., et al., A multicentre study of 513 Danish patients with systemic lupus erythematosus. II. Disease mortality and clinical factors of prognostic value. Clin Rheumatol, 1998. 17(6): p. 478-84.[5]Leffers, H.C.B., et al., Smoking associates with distinct clinical phenotypes in patients with systemic lupus erythematosus: a nationwide Danish cross-sectional study. Lupus Sci Med, 2021. 8(1).Disclosure of InterestsMartin Andersen Employee of: Novo Nordisk A/S: 2010-2014, Anders Stockmarr: None declared, Henrik Leffers: None declared, Anne Troldborg: None declared, Anne Voss: None declared, Salome Kristensen: None declared, Bent Deleuran: None declared, Lene Dreyer Speakers bureau: Speakers bureau: Eli Lilly, Galderma and Janssen, Grant/research support from: Grant from BMS outside the present work, Laura Johnsen: None declared, Ada Colic: None declared, Søren Jacobsen: None declared

Prompted by the recently reported expression of POU5F1 (OCT3/4) in epididymis, a panel of markers for carcinoma in situ (CIS) testis and testicular germ cell tumours (TGCT), including AP-2γ(TFAP2C), NANOG, OCT3/4, KIT, placental-like alkaline phosphatase (PLAP), M2A/PDPN and MAGE-A4 were examined by immunohistochemistry or in situ hybridisation in urogenital epithelia, which may interfere with detection of CIS cells in semen. In addition to OCT3/4, the expression of AP-2γ and NANOG or their variants was detected in urogenital epithelia, while other CIS markers, including PLAP/alkaline phosphatase were absent. A combination of immunocytological staining for AP-2γ or OCT3/4 and rapid cytochemical alkaline phosphatase reaction was subsequently developed. This approach was tested in 22 patients with TGCT. In 14 patients (63.6%), double stained cells were found and thus the method was proven suitable for the detection of CIS cells in semen. In conclusion, transcription factors related to pluripotency and undifferentiated state of cells, which most likely have several variants or modifications, are unexpectedly detected using currently available antibodies in urogenital epithelial cells which may be shed into semen. Combining the immunohistochemical nuclear markers with a rapid cytochemical alkaline phosphatase reaction for detection of CIS cells in ejaculates may provide a more reliable diagnostic method.

The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL mRNA and protein were found in all germ cells as well as in Sertoli and Leydig cells. EL mRNA was abundant in pre-invasive carcinoma in situ (CIS) cells and GCTs, and EL protein was present in the cytoplasm of these cells. LPL mRNA was also relatively abundant in germ cells, Sertoli cells, CIS cells and GCTs. The LPL protein, however, was restricted to the cell membranes of pachytene spermatocytes and spermatids in normal tubules, absent from CIS cells and scarcely represented in tumours. The distribution of LPL protein in non-seminomas resembled the distribution of OCT3/4, a marker of embryonal carcinoma. The results suggest that both EL and LPL participate in the supply of nutrients and steroidogenesis in the testes, and that especially EL may be important for the supply of cholesterol for testosterone production in the Leydig cells. The partial cellular separation of the expression of the two lipases in normal testis suggests the existence of distinct biological roles, perhaps developmentally regulated, as indicated by the LPL expression in GCTs with embryonic features. A high expression of EL and abundance of lipid in tubules with CIS may have a diagnostic value.

Background: A possible association between the polymorphic CAG repeat in the DNA polymerase gamma (POLG) gene and the risk of testicular germ-cell tumours (TGCT) was investigated in this study. The hypothesis was prompted by an earlier preliminary study proposing an association of the absence of the common 10-CAG-long POLG allele with testicular cancer as well as previously reported in some European populations’ association with male subfertility, which is a condition carrying an increased risk of TGCT. Patients and methods: The number of CAG repeats in both POLG alleles was established in 243 patients with TGCT and in 869 controls by the analysis of the genomic DNA fragment. Results: A significantly higher proportion of men homozygous allele of other than the common 10 CAG repeats was found among the patients with TGCT in comparison to the controls (4.9% versus 1.3%, respectively, P = 0.001). The vast majority of the homozygous patients had a seminoma (11 of 12; 97%), despite that only about half (55%) of the studied patients had this tumour type. Conclusions: The findings indicate that the POLG polymorphism may be a contributing factor in the pathogenesis of TGCT particularly in seminoma, but the mechanisms remain to be elucidated.

Udgivelsesdato: Apr Healthy men produce an enormous number of sperms, far more than necessary for conception. However, several studies suggest that semen samples where the concentration of sperms is below 40 mill/mL may be associated with longer time to pregnancy or even subfertility, and specimens where the concentration of sperms is below 15 mill/mL may carry a high risk of infertility. Historic data from the 1940s show that the bulk of young men at that time had sperm counts far above 40 mill/mL with averages higher than 100 mill/mL. However, recent surveillance studies of young men from the general populations of young men in Northern Europe show that semen quality is much poorer. In Denmark approximately 40 percent of the men have now sperm counts below 40 mill/mL. A simulation assuming that average sperm count had declined from 100 mill/mL in 'old times' to a current level close to 40 mill/mL indicated that the first decline in average sperm number of 20-40 mill/mL might not have had much effect on pregnancy rates, as the majority of men would still have had counts far above the threshold value. However, due to the assumed decline in semen quality, the sperm counts of the majority of 20 year old European men are now so low that we may be close to the crucial tipping point of 40 mill/mL spermatozoa. Consequently, we must face the possibility of more infertile couples and lower fertility rates in the future.

Recently a worldwide rise in the incidence of testicular germ cell cancer (TGCC) has been repeatedly reported. The changing disease pattern may signal that other testicular problems may also be increasing. We have reviewed recent research progress, in particular evidence gathered in the Nordic countries, which shows strong associations between testicular cancer, undescended testis, hypospadias, poor testicular development and function, and male infertility. These studies have led us to suggest the existence of a testicular dysgenesis syndrome (TDS), of which TGCC, undescended testis, hypospadias/disorders of sex differentiation and male fertility problems may be symptoms with varying penetration. In spite of their fetal origin, most of the TDS symptoms, including TGCC and poor semen quality, can only be diagnosed in adulthood. Data from a Danish-Finnish research collaboration strongly suggest that trends in TGCC rates of a population may be 'whistle blowers' of other reproductive health problems. As cancer registries are often of excellent quality - in contrast to registries for congenital abnormalities - health authorities should consider an increase in TGCC as a warning that other reproductive health problems may also be rising.

Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element in the neuroendocrine system controlling development and function of the reproductive axis; the HP unit being highly sensitive to the organizing effects of endogenous and exogenous sex steroids. To gain knowledge on the molecular mode of action and potential biomarkers of exposure to estrogenic compounds at the HP unit, we screened for differentially expressed genes at the pituitary and hypothalamus of rats after neonatal exposure to estradiol benzoate. Our analyses identified persistent up-regulation of alpha- and beta-globin mRNAs at the pituitary following neonatal estrogenization. This finding was confirmed by combination of RT-PCR analyses and in situ hybridization. Induction of alpha- and beta-globin mRNA expression at the pituitary by neonatal exposure to estrogen was demonstrated as dose-dependent and it was persistently detected up to puberty. In contrast, durable up-regulation of alpha- and beta-globin genes was not detected at the hypothalamus, cortex, cerebellum, liver and testis. Finally, enhanced levels of alpha- and beta-globin mRNAs at the pituitary were also demonstrated after neonatal administration of the anti-androgen flutamide. In summary, alpha- and beta-globin genes may prove as sensitive, pituitary-specific biomarkers of exposure to estrogenic (and/or anti-androgenic) compounds at critical periods of sex development, whose potential in the assessment of endocrine disrupting events at the HP unit merits further investigation.

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase-polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM.

Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human spermatozoa. The VD-inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression at the sperm annulus. Here, we investigated if CYP24A1 expression serves as a marker for VD metabolism in spermatozoa, and whether CYP24A1 expression was associated with semen quality. We included 130 men (53 healthy young volunteers and 77 subfertile men) for semen analysis and immunocytochemical (ICC) detection of CYP24A1. Another 40 men (22 young, 18 subfertile) were tested for in vitro effects of 1,25(OH)(2)D(3) on intracellular calcium concentration ([Ca(2+)](i)) and sperm motility. Double ICC staining showed that CYP24A1 and VDR were either concomitantly expressed or absent in 80% of the spermatozoa from young men. The median number of CYP24A1-expressing spermatozoa was 1% in subfertile men and thus significantly (p0.0005) lower than 25% in spermatozoa from young men. Moreover, CYP24A1 expression correlated positively with total sperm count, -concentration, -motility and -morphology (all p0.004), and the percentage of CYP24A1-positive spermatozoa increased (15 vs. 41%, p0.0005) after percoll-gradient-centrifugation. We noticed that the presence of3% CYP24A1-positive spermatozoa distinguished young men from subfertile men with a sensitivity of 66.0%, a specificity of 77.9% and a positive predictive value of 98.3%. Functional studies revealed that 1,25(OH)(2)D(3) increased [Ca(2+)](i) and sperm motility in young healthy men, while 1,25(OH)(2)D(3) was unable to increase motility in subfertile patients. In conclusion, we suggest that CYP24A1 expression at the annulus may serve as a novel marker of semen quality and an objective proxy for sperm function.

Prompted by the recently reported expression of POU5F1 (OCT3/4) in epididymis, a panel of markers for carcinoma in situ (CIS) testis and testicular germ cell tumours (TGCT), including AP-2γ(TFAP2C), NANOG, OCT3/4, KIT, placental-like alkaline phosphatase (PLAP), M2A/PDPN and MAGE-A4 were examined by immunohistochemistry or in situ hybridisation in urogenital epithelia, which may interfere with detection of CIS cells in semen. In addition to OCT3/4, the expression of AP-2γ and NANOG or their variants was detected in urogenital epithelia, while other CIS markers, including PLAP/alkaline phosphatase were absent. A combination of immunocytological staining for AP-2γ or OCT3/4 and rapid cytochemical alkaline phosphatase reaction was subsequently developed. This approach was tested in 22 patients with TGCT. In 14 patients (63.6%), double stained cells were found and thus the method was proven suitable for the detection of CIS cells in semen. In conclusion, transcription factors related to pluripotency and undifferentiated state of cells, which most likely have several variants or modifications, are unexpectedly detected using currently available antibodies in urogenital epithelial cells which may be shed into semen. Combining the immunohistochemical nuclear markers with a rapid cytochemical alkaline phosphatase reaction for detection of CIS cells in ejaculates may provide a more reliable diagnostic method.

The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found in other species. In mice and chicken, the lipocalin-type Pgds is specifically expressed in pre-Sertoli cells just after Sry and Sox9 and is involved in masculinisation of the developing testis. Furthermore, Pges are implicated in female reproduction including follicular development and ovulation. In this study, a sexually dimorphic expression of pgds was found in gonads of adult zebrafish with expression in testis but not in ovaries. To determine whether the sex-specific expression pattern of pgds was present in gonads of juvenile zebrafish and therefore could be an early marker of sex in zebrafish, we microdissected gonads from four randomly selected individual zebrafish for every second day in the period 2-20 days post hatch (dph) and 0-1 dph. The temporal expression of pgds and pges was investigated in the microdissected gonads, however, no differential expression that could indicate sex-specific difference between individual juvenile zebrafish was observed.

The CLLU1 expression level is a stable and inherent feature of the chronic lymphocytic leukemia clone

Large-scale methods for analysing gene expression, such as microarrays, have yielded a wealth of information about gene expression at the mRNA level. However, expression of alternative transcripts, together with the presence of a wide range of largely undescribed RNA transcripts combined with regulation from the RNA interference pathway, may cause misinterpretations when trying to base conclusions from expression data derived from studies at the mRNA level. With HLXB9, PRM1, DICER and E2F1 as examples, we here show a range of situations that can occur when investigating gene expression, and give recommendations for the complementary methods that can verify gene expression data from large-scale studies, as well as give new information regarding the regulation of specific genes. Especially, we show that the absence of a protein despite high expression of the corresponding mRNA can be caused by expression of miRNAs targeting the mRNA. Additionally, we show through cloning the presence of both known and new miRNAs in the testis emphasizing the necessity for following up mRNA expression data by investigating expression at the protein level.

Udgivelsesdato: November Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC-MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol-water-acetic acid, centrifuged, filtrated and determined by LC-MS/MS. The mean recoveries were in the range 80-110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g-4.0 ng/g.

The E2F1 transcription factor is a key regulatory factor of both apoptosis and cell cycle progression, and depending on cellular context, can act as either an oncogene or a tumour suppressor. Consequently, gene mutations that lead to deregulation of E2F1 activity are detected in a majority of human cancers.1 Recently, an array study identified E2F1 as upregulated in human testis containing preinvasive carcinoma in situ (CIS) cells when compared with normal human testis.2 Apparently, supporting this, an E2F1 overexpressing mouse demonstrated generation of CIS-like cells in testis that were otherwise atrophying owing to massive E2F1-induced apoptosis of germ cells

Background Testicular germ cell tumours (TGCTs) originate from a common precursor, carcinoma in situ (CIS). Diagnosis at the CIS stage is desirable as it minimizes the necessary treatment. A detailed clinical evaluation of an approach to detect CIS cells in the ejaculate using primordial germ cell/gonocyte markers is presented. Methods Immunocytological staining for AP-2gamma [and in some cases, OCT-3/4, NANOG or placental alkaline phosphatase (PLAP)] was performed in semen samples from 294 infertile patients and 209 patients with TGCTs or other diseases. Results Presence of AP-2gamma-stained cells was detected in 50% of participants with CIS and in 33.9% of TGCT patients before treatment (non-seminomas: 56.6%, seminomas: 17.4%). OCT-3/4 results were similar to those of AP-2gamma, whereas NANOG and PLAP stainings were unsuitable. Sensitivity was 54.5% for participants harbouring pre-invasive CIS but reduced in participants with overt TGCTs, perhaps because of obstruction. Assay specificity was 93.6%, positive predictive value (PPV) 83.3% and negative predictive value (NPV) 60.3%. Conclusions Immunocytological semen analysis based on expression of fetal germ cell markers in exfoliated cells has auxiliary diagnostic value, as it detects some patients with CIS/incipient tumour, but a negative result does not exclude TGCT. Further effort is needed to improve this assay, for example, by employing a more sensitive biochemical method of detection.

In the rat, testosterone (T) in the neonatal period plays an important role in sexual differentiation and there is a serum T surge in male rats 2 hours after birth. Pregnant female rats were exposed to various doses of bisphenol A (BPA) from gestational day 1 (GD1) through 2 hours after parturition. About half of the BPA-exposed and control dams were subjected to cesarean section on GD22. The male fetuses on GD22 were immediately sacrificed and blood was collected. The other half of the BPA-treated and control dams delivered at GD23 (parturition day). The male pups were sacrificed 2 hours after birth. Serum T concentration was determined by radioimmunoassay (RIA). The BPA concentration in the fetal serum on GD22 increased inversely to the T levels in the serum. The T concentration in the pups' serum 2 hours after birth decreased inversely to the BPA concentration in the serum. However, there were no differences in the serum T concentration among the various doses of BPA. These results suggest that exposure to BPA in utero inhibits the T surge in the neonatal period and we speculate that exposure to BPA in utero disrupts the endocrine environment in the neonatal male.

Testicular cancer is the most common malignancy among men in the reproductive age and the incidence is increasing, probably caused by environmental factors. Most testicular cancers are testicular germ cell tumours and all originate from a carcinoma in situ (CIS) pattern. In this review, we focus on the pre-invasive CIS and its possible fetal origin by reviewing recent data originating from DNA microarrays and comparative genomic hybridisations. A comparison of gene expression and genomic aberrations reveal chromosomal “hot spots” with mutual clustering of gene expression and genomic amplification. Some of the genes found in the hot spots may be involved in creating the CIS phenotype. On the other hand, many genes that are highly expressed in CIS are not present in the hot-spot areas. The gene expression profile of CIS thus most likely reflects the combined result of genomic amplification and increased transcriptional activation and/or deficiency in the epigenetic silencing of specific loci. Amplification of chromosome 12p, appears to be a good genomic marker of the transition from the pre-malignant to malignant CIS cell; this is consistent with recent findings of propagation advantages in cultured undifferentiated embryonic stem cells after spontaneous amplification in similar regions. The gene expression profile of CIS cells has remarkable similarity to that of embryonic stem cells and supports our long-standing hypothesis of an early developmental origin of CIS and testicular germ cell cancer.

It has been hypothesized that recent adverse trends in humans are linked to an increased exposure to potential endocrine disrupting agents. These include widely used compounds that mimic the action of sex hormones, including bisphenol A, phthalates and parabens. Since the chemical structure is not sufficient to determine whether a chemical will act as an oestrogen, there is a need for assays that can determine whether a compound interferes with the endocrine systems. The Environmental Protection Agency has recently suggested a testing scheme, composed of an initial screening followed by a more comprehensive investigation of chemicals that are positive in the screening. The screening will use several short-term assays to screen many thousands of compounds for potential endocrine disrupting properties. However, none of these tests determines compound-induced effects on the expression of endogenous genes, which is the cause of the adverse effects. We propose to use a precise quantification of the expression levels of endogenous oestrogen-regulated genes to test whether a chemical has oestrogenic properties, and describe how an endogenous gene expression assay can be established and conducted. Furthermore, different applications of such an assay are discussed: in cell cultures; in experimental animals; or, optimally, directly in blood samples from exposed humans.

Mutations in the androgen receptor gene are considered as incompatible with preservation of fertility and have been suggested as a cause of male infertility. Two adult brothers, referred because of gynecomastia and hormonal levels in serum indicating androgen insensitivity (high sex hormone-binding globulin, and LH levels, despite extremely high testosterone concentration), turned out to be relatives to a third young man, referred independently of the two others and exhibiting identical clinical and hormonal stigmata. In all three men, we found a C--A substitution at position 2470 (exon 7) in the androgen receptor gene, leading to a Gln824Lys mutation in the ligand-binding domain of the receptor. Exploring the family history revealed that their grandfathers, on their mothers' side, were brothers; and the Gln824Lys mutation was also found in the one of them who was still alive. Binding studies with the mutant receptor in transfected COS-7 cells, with mibolerone as ligand, exhibited equal Kd (0.7 vs. 1.0 nmol/ L), IC50 (0.8 vs. 1.1 nmol/L), and maximum binding (7.1 vs. 8.9 fmol/ 10(6) cells), as compared with the wild-type (WT) receptor. In a chloramphenicol acetyl transferase trans-activation assay, the activity of the mutant receptor was identical to that of the WT, when the synthetic androgen R1881 was'used as a ligand; but with dihydrotestosterone, in concentrations up to 10 nmol/L, the activity of Gln824Lys mutated receptor was 10-62% of the WT variant. Thus, Gln824Lys mutation was found, both in vivo and in vitro, to cause slight impairment of receptor function but was compatible with preservation of male fertility. The patients inherited the mutation from their grandfathers through their mothers, and one of the young men possessing the mutation has fathered a daughter.

Glutaryl-CoA dehydrogenase (GDH, EC 1.3.99.7) deficiency (glutaric aciduria type 1 (GA1), McKusick 231670) is an autosomal recessive disease affecting the catabolism of the amino acids lysine, hydroxylysine and tryptophan (Goodman and Frerman 1995). Patients with this disorder usually excrete high amounts of glutarate in the urine. However, patients excreting low amounts have also been reported. No correlation has been found between glutarate excretion, residual glutaryl-CoA dehydrogenase activity and severity of symptoms. The gene for glutaryl-CoA dehydrogenase has now been cloned and several mutations have been described (Goodman et al 1995). We report three families in which members had symptoms indicating glutaryl-CoA dehydrogenase deficiency but excreted no or very little free glutarate. Results of mutation analyses are presented and compared with the clinical and biochemical data.

Differential display of expressed mRNAs (DDRT-PCR) (Liang and Pardee 1992) is rapidly becoming the principal method for comparing gene expression in different cells or tissues. Differentially expressed bands can be excised from the DDRT-PCR gels, reamplified and purified (see Chap. XXVI). The next step is to identify the genes (mRNAs) that correspond to the differentially displayed bands. As a first attempt, the DNA fragment is sequenced, as described in Chap. XXXIII in this volume, followed by searches in the DNA sequence databases, using the DNA sequence as query sequence. If the gene is known, this will identify the differentially expressed gene; however, unless if human samples have been analyzed, the chance that the gene is known is rather small since relatively few nonhuman genes have been sequenced. If human samples have been analyzed, there is a high probability that the gene is represented in the databases as an expressed sequence tag (EST), and this may then identify the gene (see Chap. XXXIII). If the sequence is not present in the database, or if it is a nonhuman sample, it is necessary to clone the corresponding cDNA and sequence parts of the coding region and then repeat the search in the database with coding region sequence as query sequence(s).

The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.

TNF-α induced, in a time and dose-dependent fashion, cell-cell dissociation (dyscohesion) of endometrial epithelial cells. Within the time frame that dyscohesion was induced, TNF-α, in a dose-dependent fashion, reduced filamentous (F) actin and resulted in the loss of F-actin from the intercellular boundaries. Loss of F-actin mediated by TNF-α was not due to a reduction in the overall amount of actin or its β-isoform. Two proteins, Rho and Rho guanine nucleotide dissociation inhibitor (Rho-GDI), have been implicated in the regulation of organization of actin cytoskeleton. The reduced level of F-actin was not associated with altered expression of Rho protein, however, it was associated with an increase in the amount of Rho available for ribosylationin vitro by the C3 exoenzyme of Clostridium botulinum. The amount of Rho-GDI protein did not change after treatment with TNF-α suggesting that elevated expression of this protein is not responsible for the disassembly of actin filaments. These findings show that TNF-α induces dyscohesion. Dyscohesion induced by this cytokine is associated with perturbation of the actin cytoskeleton which may be due to the regulatory role of TNF-α on Rho.

We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

The sequence of the 16S rRNA gene from the archaebacterium Halococcus morrhua was determined by the dideoxynucleotide sequencing method. It is 1475 nucleotides long. This is the second archaebacterial sequence to be determined and it provides sequence comparison evidence for the secondary structural elements confined to the RNAs of this kingdom and, also, support for controversial or additional base pairing in the eubacterial RNAs. Six structural features are localized that have varied during the evolution of the archaebacteria, eubacteria and eukaryotes. Moreover, although the secondary structures of both sequenced archaebacterial RNAs strongly resemble those of eubacteria, they contain sufficient eukaryotic-like structural characteristics to reinforce the view that they belong to a separate line of evolutionary descent.

The 23 S RNA genes representative of each of the main archaebacterial subkingdoms, Desulfurococcus mobilis an extreme thermophile, Halococcus morrhuae an extreme halophile and Methanobacterium thermoautotrophicum a thermophilic methanogen, were cloned and sequenced. The inferred RNA sequences were aligned with all the available 23 S-like RNAs of other archaebacteria, eubacteria/chloroplasts and the cytoplasm of eukaryotes. Universal secondary structural models containing six major structural domains were refined, and extended, using the sequence comparison approach. Much of the present structure was confirmed but six new helices were added, including one that also exists in the eukaryotic 5.8 S RNA, and extensions were made to several existing helices. The data throw doubt on whether the 5' and 3' ends of the 23 S RNA interact, since no stable helix can form in either the extreme thermophile or the methanogen RNA. A few secondary structural features, specific to the archaebacterial RNAs were identified; two of these were supported by a comparison of the archaebacterial RNA sequences, and experimentally, using chemical and ribonuclease probes. Seven tertiary structural interactions, common to all 23 S-like RNAs, were predicted within unpaired regions of the secondary structural model on the basis of co-variation of nucleotide pairs; two lie in the region of the 23 S RNA corresponding to 5.8 S RNA but they are not conserved in the latter. The flanking sequences of each of the RNAs could base-pair to form long RNA processing stems. They were not conserved in sequence but each exhibited a secondary structural feature that is common to all the archaebacterial stems for both 16 S and 23 S RNAs and constitutes a processing site. Kingdom-specific nucleotides have been identified that are associated with antibiotic binding sites at functional centres in 23 S-like RNAs: in the peptidyl transferase centre (erythromycin-domain V) the archaebacterial RNAs classify with the eukaryotic RNAs; at the elongation factor-dependent GTPase centre (thiostrepton-domain II) they fall with the eubacteria, and at the putative amino acyl tRNA site (alpha-sarcin-domain VI) they resemble eukaryotes. Two of the proposed tertiary interactions offer a structural explanation for how functional coupling of domains II and V occurs at the peptidyl transferase centre. Phylogenetic trees were constructed for the archaebacterial kingdom, and for the other two kingdoms, on the basis of the aligned 23 S-like RNA sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system. Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis. The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data. Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits. Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop. This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site. Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding. Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV. Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites. alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain. Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.

Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks to the advent of microsequencing the databases make it possible to directly link protein and DNA information.

Domain I of 23 S RNA of Escherichia coli was probed in renatured RNA, in the protein L24-RNA complex and in 50 S subunits with ribonucleases specific for single- and double-stranded regions and with chemical reagents specific for guanosines (N-1 and N-2), adenosines (N-1, N-7 and N-6), cytidines (N-3) and uridines (N-3). Reactive sites were detected by a reverse transcriptase procedure. The results support most new features of the latest version of the Santa Cruz/Urbana model of the secondary structure, which is based on evidence from sequence comparison. Most double-helical segments were reactive to cobra venom ribonuclease to some degree; the exceptions were the five "long-range" helices that are probably compactly folded within the structure. The data provide evidence for the occurrence of A(syn) X G(anti) pairings in internal loops and at the ends of some helices; they also support the existence of extensive higher-order structuring, especially within the interhelical regions, and are compatible with two of three tertiary interactions in the free RNA that were predicted from comparative sequence studies. Protein L24 is the only primary binding protein that associates with domain I and it strongly protects two sites against ribonuclease and chemical activity. Site A has the properties of a classic protein binding site and we conclude from four lines of evidence that it is the primary attachment site. Site B is rich in highly conserved, unpaired adenosine residues and lies in a potentially critical region of the structure adjoining a group of long-range helices; we infer that L24 binding here is related to the important role of L24 in initiating ribosomal assembly; the existence of both sites is supported, independently, by genetic experiments. L24-induced enhanced reactivities were detected throughout the domain and are consistent with a general "tuning" of the RNA structure. The RNA domain in the 50 S subunits is almost completely resistant to ribonucleases and only a few sites, mainly interhelical, are accessible to chemical reagents. The appearance of several newly reactive nucleotides in the subunit RNA and the enhancement of some others suggest that some minor conformational changes occur on assembly. Nevertheless, the minimal secondary structure of the renatured RNA appears to be retained. We draw the general conclusion that domain I is a highly structured domain that is important for initiating assembly and for the subsequent organization of the ribosome.

We describe, for the first time, an intron that is located in the variable loop of a tRNA. It is 18 nucleotides long and occurs within a precursor tRNAGly from the extreme thermophile Thermofilum pendens. The variable loop is less critical for tRNA function than the anticodon arm where other class III introns occur. This raises the possibility that tRNAs with a large variable loop (type II) arose from those with a small variable loop (type I) by retaining a splicing-deficient intron.

LANCL1 (LanC-like protein 1) is related to the bacterial LanC (lanthionine synthetase C) family, which is involved in the biosynthesis of antimicrobial peptides. Highest expression levels of LANCL1 are found in testes and brain, two organs that exist behind blood-tissue barriers. In the mouse, the establishment of an impermeable blood-testis barrier occurs between day 10-16 post natal (pn). Differential display analysis showed that the expression level of LANCL1 mRNA in mouse testes was very low until day 16 pn, but increased gradually from day 16 pn to reach a maximum on days 22- 24 pn followed by a slight reduction from day 26 pn to adult animals. Thus, the expression of LANCL1 mRNA is initiated following the establishment of the blood-testis barrier. In situ hybridisation revealed that LANCL1 mRNA was induced in diplotene spermatocytes, which appear for the first time in mouse testes between days 18 and 20 pn, verifying the expression profile determined by differential display. LANCL1 mRNA level remained high in the meiotic division phase and in early round spermatids, but was down regulated in elongating spermatids and it was undetectable in step 9 elongating spermatids in stage IX (as defined by Russel et al., 1990). The steady decrease in expression level from round spermatids in stage I to elongating spermatids in stage IX suggested that LANCL1 mRNA was not transcribed in spermatids. LANCL1 expression in rat testes was initiated already in pachytene spermatocytes in stage IX, but otherwise similar to mouse.

Background Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation. Methods The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads. Results The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling. Conclusion The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.