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Objective: To evaluate the risk of inducing RhD immunisation in pregnancies of RhD-negative mothers with an RhD-positive fetus undergoing chorionic villus samplings (CVS) or amniocenteses (AC).Design, Setting and Population: Registry-based study in a Danish cohort which has not been given rhesus prophylaxis.Methods: Data were retrieved from the Department of Clinical Immunology at Rigshospitalet. All RhD-negative women carrying an RhD-positive fetus with screen test results from weeks 8-12 and weeks 25-29 were linked to data from the Danish Fetal Medicine Database. Data were divided into cases where no invasive prenatal diagnostic procedure was performed, cases that had AC performed, and cases that had CVS performed.Main Outcome Measures: A comparison of the proportion of women who developed RhD immunisation between the two screen tests.Results: The cohort consisted of 10 085 women: 9353 had no invasive procedures performed, 189 had AC and 543 had CVS performed. No women were immunised spontaneously or due to the procedure between the first and second screen test in the group with no procedure performed, or in the AC group. One woman was immunised in the CVS group. When comparing the proportion of women who was immunised in the CVS group with the no invasive test group a non-significant difference was found (P = 0.055).Conclusion: The RhD immunisation rate before gestational weeks 25-29 in RhD-negative women carrying an RhD-positive fetus is very low, even in women undergoing prenatal invasive testing without rhesus prophylaxis.Tweetable Abstract: The RhD immunisation rate during pregnancy is very low even in women undergoing prenatal invasive testing. [ABSTRACT FROM AUTHOR]

ABSTRACT Objective Prenatal and postnatal RhD prophylaxis reduces the risk of RhD immunization in pregnancies of RhD-negative women. Based on the result from prenatal screening for the fetal RHD gene, prenatal RhD prophylaxis in Denmark is targeted to RhD-negative women who carry an RhD-positive fetus. Here, we present a 2-year evaluation of a nationwide prenatal RHD screening. Methods Blood samples were drawn from RhD-negative women in gestational week 25. DNA was extracted from maternal plasma and analyzed for the RHD gene. The prenatal RHD results were compared with the serological typing of newborns in 12,668 pregnancies. Early compliance was assessed for 690 pregnancies. Results The sensitivity for the detection of fetal RHD was 99.9% (95% CI: 99.7-99.9%). Unnecessary recommendation of prenatal RhD prophylaxis was avoided in 97.3% of the women carrying an RhD-negative fetus. Fetuses that were seropositive for RhD were not detected in 11 pregnancies (0.087%). The sample uptake percentage was 84.2%, and the compliance for prenatal anti-D administration was 93.2%. Conclusion The high sensitivity, maintained over 2 years, underlines the reliability of routine prenatal fetal RHD screening in RhD-negative pregnant women, specifically at 25 weeks of gestation. The remaining challenges are logistical and are related to program compliance. © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background As of January 1, 2010, Denmark implemented a national antenatal directed anti-D prophylaxis program. Prior to this, only postnatal prophylaxis directed by serology of cord blood was practiced. A preparatory work for modernization and optimization of fetal care was carried out by the National Board of Health with participation of leading obstetricians, specialists in fetal medicine, general practitioners, midwives, clinical immunologists and administrators. The promising results of non-invasive prenatal diagnosis based on cell free fetal DNA enabled an antenatal directed use of anti-D to those with a D positive fetus, thus eliminating approximately 40% of the antenatal use of anti-D. The complete program now includes an antenatal RHD screening in gestational week 25, and a postnatal serological RhD typing of cord blood, in both instances followed by administration of anti-D to those found positive. Aim The explicit aim of the RHD screening was to obtain the highest possible sensitivity to recommend anti-D in all cases where it might be indicated whereas specificity was of secondary priority. Methods Health services in Denmark are organized in five regions; accordingly screening has been implemented in every region, each using its own in-house screening methodology. However, all regions have implemented automated DNA purification and the assays are variations over a theme of multiple PCRs covering several exons. A control reaction for DNA purification is obligatory. However, a control for fetal DNA is not included. A working group with members from all regions has implemented a national scheme for quality control. Digital on-line delivery of results to general practitioners, midwives and obstetricians is implemented in most regions thus cutting down on cost and time. Results The continuation of postnatal serology has allowed a quality control of RHD screening. During the first six months of screening only two false negative results occurred out of 2312 tested (0·087%) suggesting a sensitivity of 99·9%. Due to the sacrifice of specificity some unnecessary use of anti-D occurred, 39 cases or 1·7%. Discussion The elimination of 38·3% of the use of anti-D, that is unnecessary and the related clinical contacts balances the expenses for the screening. An unforeseen benefit of the screening has emerged. The availability of the antenatal RHD results at the time of delivery has simplified logistics and preliminary clinical action is taken based on the antenatal result. Only in the very rare occasions of a (false negative) discrepancy between antenatal and postnatal results is communication needed between laboratory, clinician, and patient. The challenge will be to maintain the high standards in a possible future setting without serology serving as a benchmark for quality control. [ABSTRACT FROM AUTHOR]

Interferon-γ secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human leucocyte antigen (HLA) class I binders (KD ≤ 5 nM). However, five of the individuals tested did not show typical CD8+ T cell-mediated HLA class I-restricted responses. Instead, these donors showed CD4+ T cell-dependent responses against four of a total of eight antigenic 9-mer peptides discovered recently by our group. These latter responses were blocked specifically in the presence of anti-HLA class II antibody. We conclude that long-lived memory responses against pox virus-derived 9-mer peptides, with high binding affinity for HLA class I molecules, are mediated in some cases by CD4+ T cells and apparently restricted by HLA class II molecules. [ABSTRACT FROM AUTHOR]

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen–receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1–10 nM 17 β-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17 β-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17 β-estradiol (0.001–10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER- α is expressed in osteoclast precursors, whereas ER- β is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER- α for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts. [ABSTRACT FROM AUTHOR]

The soluble amphiphilic glycoprotein, Ag1 (gp60), purified from supernatants of in vitro cultures of Plasmodium falciparum has a molecular mass of 60 kDa and did not exhibit size variation in the different P. falciparum isolates tested by immunoblotting. Ag1 was shown to interact with the lectin Erythrina christagalli agglutinin, which is specific for carbohydrates bearing β- d-galactose(1-4)- d− N-acetylglucosamine. Indirect immunofluorescence studies showed that Ag1 is located on the surface of trophozoites and schizonts but not on the surface of merozoites. Ag1 is recognized by human immune sera from six different malaria-endemic regions. Ag1 induces in vitro proliferation of lymphocytes from malaria-immune individuals in an antigenspecific manner. [ABSTRACT FROM AUTHOR]

Abstract Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay. Results Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of parasite counts through CMXRos staining and flow cytometry. Conclusions A flow cytometry method based on CMXRos staining for detection of live parasite populations has been optimized. This is a rapid and sensitive method with high inter-assay reproducibility which can reliably determine the anti-parasite effect mediated by antibodies in functional in vitro assays such as ADCI assay.

Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours). The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

Abstract Background Normal osteoclasts resorb bone by secretion of acid and proteases. Recent studies of patients with loss of function mutations affecting either of these processes have indicated a divergence in osteoclastic phenotypes. These difference in osteoclast phenotypes may directly or indirectly have secondary effects on bone remodeling, a process which is of importance for the pathogenesis of both osteoporosis and osteoarthritis. We treated human osteoclasts with different inhibitors and characterized their resulting function. Methods Human CD14 + monocytes were differentiated into mature osteoclasts using RANKL and M-CSF. The osteoclasts were cultured on bone in the presence or absence of various inhibitors: Inhibitors of acidification (bafilomycin A1, diphyllin, ethoxyzolamide), inhibitors of proteolysis (E64, GM6001), or a bisphosphonate (ibandronate). Osteoclast numbers and bone resorption were monitored by measurements of TRACP activity, the release of calcium, CTX-I and ICTP, as well as by counting resorption pits. Results All inhibitors of acidification were equally potent with respect to inhibition of both organic and inorganic resorption. In contrast, inhibition of proteolysis by E64 potently reduced organic resorption, but only modestly suppressed inorganic resorption. GM6001 alone did not greatly affect bone resorption. However, when GM6001 and E64 were combined, a complete abrogation of organic bone resorption was observed, without a great effect on inorganic resorption. Ibandronate abrogated both organic and inorganic resorption at all concentrations tested [0.3-100 μM], however, this treatment dramatically reduced TRACP activity. Conclusions We present evidence highlighting important differences with respect to osteoclast function, when comparing the different types of osteoclast inhibitors. Each class of osteoclast inhibitors will lead to different alterations in osteoclast quality, which secondarily may lead to different bone qualities.

Background: A good educational climate is essential for delivering high-quality training for medical trainees, professional development, and patient care. The aim of this study was to (1) validate the Dutch Residency Educational Climate Test (D-RECT) in a Danish setting and (2) describe and evaluate the educational climate among medical trainees., Methods: D-RECT was adopted in a three-step process: translation of D-RECT into Danish (DK-RECT), psychometric validation, and evaluation of educational climate. Trainees from 31 medical specialties at Copenhagen University Hospital - Rigshospitalet, Denmark were asked to complete an online survey in a cross-sectional study., Results: We performed a forward-backward translation from Dutch to Danish. Confirmatory factor analysis showed that DK-RECT was robust and valid. The reliability analysis showed that only seven trainees from one specialty were needed for a reliable result. With 304 trainees completing DK-RECT, the response rate was 68%. The subsequent analysis indicated a positive overall educational climate, with a median score of 4.0 (interquartile range (IQR): 3.0-5.0) on a five-point Likert scale. Analysis of the subscales showed that the subscale Feedback received the lowest ratings, while Supervision and Peer collaboration were evaluated highest., Conclusions: Psychometric validation of D-RECT in a Danish context demonstrated valid results on the educational climate in specialist training. DK-RECT can be used to evaluate the effectiveness of interventions in the future and can facilitate the conversation on the educational climate., (© 2023. The Author(s).)

Background: Whether natural selection may have attributed to the observed blood group frequency differences between populations remains debatable. The ABO system has been associated with several diseases and recently also with susceptibility to COVID-19 infection. Associative studies of the RhD system and diseases are sparser. A large disease-wide risk analysis may further elucidate the relationship between the ABO/RhD blood groups and disease incidence., Methods: We performed a systematic log-linear quasi-Poisson regression analysis of the ABO/RhD blood groups across 1,312 phecode diagnoses. Unlike prior studies, we determined the incidence rate ratio for each individual ABO blood group relative to all other ABO blood groups as opposed to using blood group O as the reference. Moreover, we used up to 41 years of nationwide Danish follow-up data, and a disease categorization scheme specifically developed for diagnosis-wide analysis. Further, we determined associations between the ABO/RhD blood groups and the age at the first diagnosis. Estimates were adjusted for multiple testing., Results: The retrospective cohort included 482,914 Danish patients (60.4% females). The incidence rate ratios (IRRs) of 101 phecodes were found statistically significant between the ABO blood groups, while the IRRs of 28 phecodes were found statistically significant for the RhD blood group. The associations included cancers and musculoskeletal-, genitourinary-, endocrinal-, infectious-, cardiovascular-, and gastrointestinal diseases., Conclusions: We found associations of disease-wide susceptibility differences between the blood groups of the ABO and RhD systems, including cancer of the tongue, monocytic leukemia, cervical cancer, osteoarthrosis, asthma, and HIV- and hepatitis B infection. We found marginal evidence of associations between the blood groups and the age at first diagnosis., Funding: Novo Nordisk Foundation and the Innovation Fund Denmark., Competing Interests: PB, MH, KB No competing interests declared, PJ has received grants from the AP Møller Foundation, Innovation Fund Denmark and Novo Nordisk Foundation. The author has been issued the following patents: Publication no: 20110201553, 20110268732, 20130040898, 20130261177, 20150057325, 20160113891, 9381166, 9381243, 20160250164, 9433589, 20160303040 and US20090053193A1. PI Johansson reports ownership of stocks in Trial-Lab AB, Endothel Pharma ApS, TissueLink ApS, and MoxieLab ApS. PI Johansson declares that the financial interests listed have no impact on the submitted work. The author has no other competing interests to declare. The author declares that the financial interests listed have no impact on the submitted work, SB participates on the Danish National Genome Center advisory board and is the Chairman for the data infrastructure board. The author has stock in Intomics A/S, Hoba Therapeutics Aps, Novo Nordisk A/S, Lundbeck A/S and ALK Abello. The author participates on the board of directors for both Proscion A/S and Intomics A/S. The author has no other competing interests to declare. SB declares that the financial interests listed have no impact on the submitted work, (© 2023, Bruun-Rasmussen et al.)