Anandamide modulation of monocyte-derived Langerhans cells: implications for immune homeostasis and skin inflammation.

Introduction: The endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against pathogens and are essential for the skin's specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids on these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation. Methods: Human monocytes were differentiated into moLCs using established protocols. Anandamide was applied during the differentiation process to test its effect on the viability, marker expression, and cytokine production of the cells, as well as in short term treatments for intracellular calcium measurement. TLR ligands applied after the differentiation protocol were used to activate moLCs. The impact of anandamide on the functionality of moLCs was further assessed using differential gene expression analysis of bulk RNA-Seq data, moLC-T cell cocultures, while ELISpot was employed to determine polarization of T cells activated in the aforementioned cocultures. Results: Anandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. MoLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNA sequencing analysis of moLCs differentiated with anandamide showed modest changes compared to control cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype. Discussion: Our results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.

Magneto-optical assessment of Plasmodium parasite growth via hemozoin crystal size.

Hemozoin is a natural biomarker formed during the hemoglobin metabolism of Plasmodium parasites, the causative agents of malaria. The rotating-crystal magneto-optical detection (RMOD) has been developed for its rapid and sensitive detection both in cell cultures and patient samples. In the current article we demonstrate that, besides quantifying the overall concentration of hemozoin produced by the parasites, RMOD can also track the size distribution of the hemozoin crystals. We establish the relations between the magneto-optical signal, the mean parasite age and the median crystal size throughout one erythrocytic cycle of Plasmodium falciparum parasites, where the latter two are determined by optical and scanning electron microscopy, respectively. The significant correlation between the magneto-optical signal and the stage distribution of the parasites indicates that the RMOD method can be utilized for species-specific malaria diagnosis and for the quick assessment of drug efficacy.

Rhoptry secretion system structure and priming in Plasmodium falciparum revealed using in situ cryo-electron tomography.

Apicomplexan parasites secrete contents of the rhoptries, club-shaped organelles in the apical region, into host cells to permit their invasion and establishment of infection. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is unknown whether a similar molecular machinery exists in the malaria parasite Plasmodium. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct apical vesicle docking the tips of the two rhoptries to the RSA. We also discover two additional rhoptry organizations that lack the apical vesicle. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to different rhoptry organizations. Our results highlight previously unknown steps in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

External quality assessment providers' services appear to more impact the immunohaematology performance of laboratories than national regulatory and economic conditions.

OBJECTIVES: Medical laboratories may, at their own discretion, exceed but not undercut regulatory quality requirements. Available economic resources, however, may drive or hinder eagerness to exceed minimum requirements. Depending on the respective scopes of regulatory and economic framework conditions, differing levels of quality efforts to safeguard laboratory performance can be anticipated. However, this has not yet been investigated. METHODS: Immunohaematology external quality assessment (EQA) results collected by 26 EQA providers from their participant laboratories in 73 countries from 2004 to 2019 were evaluated. Error rates were aggregated in groups according to the respective national regulatory and economic framework conditions, to whether or not expert advice was provided in case of incorrect results, and the frequency of EQA samples. RESULTS: These representative data indicate no association between national regulatory (mandatory participation in EQA, monitoring of performance of individual laboratories by authorities, financial consequences of incorrect results) and economic (level of national income, share of national health expenditure) conditions to the quality performance of medical laboratories in immunohaematology. However, EQA providers' support for laboratories in the event of incorrect results appear to be associated with lower error rates, but a high EQA sample frequency with higher error rates. CONCLUSIONS: Further research into the impact of introducing or changing services of EQA providers is needed to confirm the results found in this first of its kind study.

Rapid and quantitative antimalarial drug efficacy testing via the magneto-optical detection of hemozoin.

Emergence of resistant Plasmodium species makes drug efficacy testing a crucial part of malaria control. Here we describe a novel assay for sensitive, fast and simple drug screening via the magneto-optical detection of hemozoin, a natural biomarker formed during the hemoglobin metabolism of Plasmodium species. By quantifying hemozoin production over the intraerythrocytic cycle, we reveal that hemozoin formation is already initiated by ~ 6-12 h old ring-stage parasites. We demonstrate that the new assay is capable of drug efficacy testing with incubation times as short as 6-10 h, using synchronized P. falciparum 3D7 cultures incubated with chloroquine, piperaquine and dihydroartemisinin. The determined 50% inhibitory concentrations agree well with values established by standard assays requiring significantly longer testing time. Accordingly, we conclude that magneto-optical hemozoin detection provides a practical approach for the quick assessment of drug effect with short incubation times, which may also facilitate stage-specific assessment of drug inhibitory effects.

Highly Sensitive and Rapid Characterization of the Development of Synchronized Blood Stage Malaria Parasites Via Magneto-Optical Hemozoin Quantification.

The rotating-crystal magneto-optical diagnostic (RMOD) technique was developed as a sensitive and rapid platform for malaria diagnosis. Herein, we report a detailed in vivo assessment of the synchronized Plasmodium vinckei lentum strain blood-stage infections by the RMOD method and comparing the results to the unsynchronized Plasmodium yoelii 17X-NL (non-lethal) infections. Furthermore, we assess the hemozoin production and clearance dynamics in chloroquine-treated compared to untreated self-resolving infections by RMOD. The findings of the study suggest that the RMOD signal is directly proportional to the hemozoin content and closely follows the actual parasitemia level. The lack of long-term accumulation of hemozoin in peripheral blood implies a dynamic equilibrium between the hemozoin production rate of the parasites and the immune system's clearing mechanism. Using parasites with synchronous blood stage cycle, which resemble human malaria parasite infections with Plasmodium falciparum and Plasmodium vivax, we are demonstrating that the RMOD detects both hemozoin production and clearance rates with high sensitivity and temporal resolution. Thus, RMOD technique offers a quantitative tool to follow the maturation of the malaria parasites even on sub-cycle timescales.

Uracil moieties in Plasmodium falciparum genomic DNA.

Plasmodium falciparum parasites undergo multiple genome duplication events during their development. Within the intraerythrocytic stages, parasites encounter an oxidative environment and DNA synthesis necessarily proceeds under these circumstances. In addition to these conditions, the extreme AT bias of the P. falciparum genome poses further constraints for DNA synthesis. Taken together, these circumstances may allow appearance of damaged bases in the Plasmodium DNA. Here, we focus on uracil that may arise in DNA either via oxidative deamination or thymine-replacing incorporation. We determine the level of uracil at the ring, trophozoite, and schizont intraerythrocytic stages and evaluate the base-excision repair potential of P. falciparum to deal with uracil-DNA repair. We find approximately 7-10 uracil per million bases in the different parasite stages. This level is considerably higher than found in other wild-type organisms from bacteria to mammalian species. Based on a systematic assessment of P. falciparum genome and transcriptome databases, we conclude that uracil-DNA repair relies on one single uracil-DNA glycosylase and proceeds through the long-patch base-excision repair route. Although potentially efficient, the repair route still leaves considerable level of uracils in parasite DNA, which may contribute to mutation rates in P. falciparum.

[Areas and challenges of oncopsychological rehabilitation]. / Az onkopszichológiai rehabilitáció területei és kihívásai.

Patients with cancer present a number of different difficulties that adversely affect their health care and recovery (e.g. poor communication with physicians, lack of knowledge about their illness and its management, financial problems). Furthermore, mental health problems, such as distress, depression and anxiety, are common amongst patients with cancer. These mental health problems are additional contributors to functional impairment in carrying out family, work, and other social roles, poor adherence to medical treatments, and adverse medical outcomes. Oncopsychosocial rehabilitation aims to optimize the possibilities of medical health care through psychological interventions by helping cancer patients and their families and/or health care workers with the management of the psychological and social aspects of the illness. Oncopsychosocial rehabilitation includes all psychosocial interventions that are designed to positively influence patient psychosocial adaptation and adjustment to diagnosis, treatment, and survivorship. Oncopsychological rehabilitation could also manage cancer related distress and other psychosocial problems with specific types of treatments or interventions including prevention, relaxation techniques, structured psychoeducational interventions including sexual information and/or preparation for surgery, and various methods of psychotherapy.

Efficient monitoring of the blood-stage infection in a malaria rodent model by the rotating-crystal magneto-optical method.

Intense research efforts have been focused on the improvement of the efficiency and sensitivity of malaria diagnostics, especially in resource-limited settings for the detection of asymptomatic infections. Our recently developed magneto-optical (MO) method allows the accurate quantification of malaria pigment crystals (hemozoin) in blood by their magnetically induced rotation. First evaluations of the method using ß-hematin crystals and in vitro P. falciparum cultures implied its potential for high-sensitivity malaria diagnosis. To further investigate this potential, here we study the performance of the method in monitoring the in vivo onset and progression of the blood-stage infection in a rodent malaria model. Our results show that the MO method can detect the first generation of intraerythrocytic P. berghei parasites 66-76 hours after sporozoite injection, demonstrating similar sensitivity to Giesma-stained light microscopy and exceeding that of flow cytometric techniques. Magneto-optical measurements performed during and after the treatment of P. berghei infections revealed that both the follow up under treatment and the detection of later reinfections are feasible with this new technique. The present study demonstrates that the MO method - besides being label and reagent-free, automated and rapid - has a high in vivo sensitivity and is ready for in-field evaluation.

The use of microdialysis techniques in mice to study P-gp function at the blood-brain barrier.

An integrated assay system involving dual/triple-probe microdialysis techniques in rats was developed earlier for testing interactions with P-glycoprotein (P-gp) at the blood-brain barrier using quinidine/PSC-833 as a P-gp substrate/inhibitor combination. The aim of the present study was to expand our assay system to mice using microdialysis with simultaneous sampling of blood and brain and to compare the result with a primary mouse brain endothelial cell monolayer (pMBMEC) assay. Brain penetration of quinidine was dose dependent in both anesthetized and awake mice after intraperitoneal drug administration. PSC-833 pretreatment caused a 2.5- to 3.4-fold increase in quinidine levels of brain dialysate samples in anesthetized or awake animals, after single or repeated administration of PSC-833. In pMBMEC, a 2.0- to 2.5-fold efflux ratio was observed in the transcellular transport of quinidine. The P-gp-mediated vectorial transport of quinidine was eliminated by PSC-833. These results indicate that quinidine with PSC-833 is a good probe substrate-reference inhibitor combination for testing drug-drug interactions with P-gp in the in vivo and in vitro mouse systems. With increasing number of humanized transgenic mice, a test system with mouse microdialysis experimentation becomes more important to predict drug-drug interactions in humans.

Quinidine as an ABCB1 probe for testing drug interactions at the blood-brain barrier: an in vitro in vivo correlation study.

This study provides evidence that quinidine can be used as a probe substrate for ABCB1 in multiple experimental systems both in vitro and in vivo relevant to the blood-brain barrier (BBB). The combination of quinidine and PSC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on ABCB1. Effects of quinidine and substrate-inhibitor interactions were tested in a membrane assay and in monolayer assays. The authors compared quinidine and digoxin as ABCB1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in ATPase and monolayer efflux assays employing MDCKII-MDR1 and the rat brain microcapillary endothelial cell system. Brain exposure to quinidine was tested in dual-/triple-probe microdialysis experiments in rats by assessing levels of quinidine in blood and brain. Comparing quinidine levels in dialysate samples from valspodar-treated and control animals, it is evident that systemic/local administration of the inhibitor diminishes the pumping function of ABCB1 at the BBB, resulting in an increased brain penetration of quinidine. In sum, quinidine is a good probe to study ABCB1 function at the BBB. Moreover, quinidine/PSC-833 is an ABCB1-specific substrate/inhibitor combination applicable to many assay systems both in vitro and in vivo.

Extramasticatory dental wear reflecting habitual behavior and health in past populations.

In skeletal remains, teeth are valuable sources of information regarding age, diet, and health. Dental wear is especially helpful in reconstructions of dietary patterns in populations of varying subsistence. In past societies, teeth have also been used as "a third hand" or as a "tool." The present article examines this type of dental wear and traits attributed to habitual behavior during prehistoric and historic times. Terminology and classification of habitual dental wear are described mainly by appearance, for instance, notching, grooving, cuts, scrapes, and polished surfaces, and their characteristics are illuminated by different case studies. Secondary health effects caused by the extramasticatory use of teeth, such as periapical lesions, tilting, skeletal changes at the temporomandibular joint, chipping, and antemortem tooth loss are also examined. During the examination of extramasticatory dental wear, information should be recorded on morphology, size, frequency, intensity, and location within the dental arch, as well as descriptions and detailed photographic documentation. The advantage of using a low- to medium-resolution microscope in all dental examination is emphasized. By categorizing the wear marks, characteristics are emphasized rather than an exact causing agent. In this way, tentative analogies for the origin of different extramasticatory wear, and consequently for human behavior in the past, can be avoided.

Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry.

BACKGROUND: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. METHODS: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs+. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [2H12]-Cyclosporin A [13C, 2H3]-Rapamycin, [13C, 2H2]-Tacrolimus, and [13C2, 2H4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. RESULTS: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8 - 102.5% for cyclosporin A, 101.6 - 103.0% for sirolimus, 100.0 - 101.2% for tacrolimus, and 99.5 - 102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17 - 2.60% for cyclosporin A in the concentration range between 8.1 and 979 microg/L, 0.92 - 1.72% for sirolimus in the concentration range between 2.1 and 33.2 microg/L, 0.44 - 1.06% for tacrolimus in the concentration range between 2.0 and 30.8 microg/L and 0.82 - 4.34% for everolimus in the concentration range between 2.1 and 31.4 microg/L. CONCLUSIONS: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.

High frequency of lactose intolerance in a prehistoric hunter-gatherer population in northern Europe.

BACKGROUND: Genes and culture are believed to interact, but it has been difficult to find direct evidence for the process. One candidate example that has been put forward is lactase persistence in adulthood, i.e. the ability to continue digesting the milk sugar lactose after childhood, facilitating the consumption of raw milk. This genetic trait is believed to have evolved within a short time period and to be related with the emergence of sedentary agriculture. RESULTS: Here we investigate the frequency of an allele (-13910*T) associated with lactase persistence in a Neolithic Scandinavian population. From the 14 individuals originally examined, 10 yielded reliable results. We find that the T allele frequency was very low (5%) in this Middle Neolithic hunter-gatherer population, and that the frequency is dramatically different from the extant Swedish population (74%). CONCLUSIONS: We conclude that this difference in frequency could not have arisen by genetic drift and is either due to selection or, more likely, replacement of hunter-gatherer populations by sedentary agriculturalists.

Ancient DNA reveals lack of continuity between neolithic hunter-gatherers and contemporary Scandinavians.

The driving force behind the transition from a foraging to a farming lifestyle in prehistoric Europe (Neolithization) has been debated for more than a century [1-3]. Of particular interest is whether population replacement or cultural exchange was responsible [3-5]. Scandinavia holds a unique place in this debate, for it maintained one of the last major hunter-gatherer complexes in Neolithic Europe, the Pitted Ware culture [6]. Intriguingly, these late hunter-gatherers existed in parallel to early farmers for more than a millennium before they vanished some 4,000 years ago [7, 8]. The prolonged coexistence of the two cultures in Scandinavia has been cited as an argument against population replacement between the Mesolithic and the present [7, 8]. Through analysis of DNA extracted from ancient Scandinavian human remains, we show that people of the Pitted Ware culture were not the direct ancestors of modern Scandinavians (including the Saami people of northern Scandinavia) but are more closely related to contemporary populations of the eastern Baltic region. Our findings support hypotheses arising from archaeological analyses that propose a Neolithic or post-Neolithic population replacement in Scandinavia [7]. Furthermore, our data are consistent with the view that the eastern Baltic represents a genetic refugia for some of the European hunter-gatherer populations.

Dental wear and oral pathology: possible evidence and consequences of habitual use of teeth in a Swedish Neolithic sample.

Atypical wear and oral lesions were studied in the dental remains from the Middle Neolithic Pitted Ware Culture site Ajvide on the island of Gotland in the Baltic Sea. Teeth (n = 764) from 17 males and 11 females were examined microscopically to document unusual wear, assess wear patterns and oral lesions as evidence of habitual behavior, and determine their effect on the oral health of this population. Five atypical wear types were observed: occlusal facets, occlusal excessive load, labial vertical striae, labial horizontal striae, and interproximal striae. Three oral lesions were recorded: chipping, periapical lesions, and dental tilting. Results indicate that teeth were used in a habitual manner at Ajvide, based on the morphology and regularity of the patterns of atypical wear. Differences were observed between the sexes, indicating gender-related differences in the habitual use of teeth. Some wear categories showed a significant correlation with age, signifying increased or accumulated wear with age. Statistically significant positive correlations were found in the molars between occlusal excessive load wear and periapical lesions as well as tilting. Other apparent links were also observed between chipping and vertical striae as well as excessive load, although these were not significant. This suggests a relationship between dental wear and dental pathologies at this site, suggesting that habitual use of teeth indirectly affected the general oral health at the site. Wear patterns, furthermore, seem to mirror both frequent activities as well as single events.

Modified HPLC-electrospray ionization/mass spectrometry method for HbA1c based on IFCC reference measurement procedure.

BACKGROUND: Monitoring of hemoglobin A(1c) (HbA(1c)) is important in the management of diabetes. The IFCC reference measurement procedure for HbA(1c) is based on the ratio of glycated to nonglycated N-terminal hexapeptides of the beta-chains of hemoglobin after digestion with Glu-C endoproteinase. We developed a modification of the original reference measurement procedure with HPLC-electrospray ionization/mass spectrometry (ESI/MS). METHOD: We performed chromatographic separation of the hexapeptides using a C12 reversed-phase column and a binary gradient system consisting of a mixture of H(2)O/acetonitrile/formic acid. RESULTS: Using this method, we obtained higher signal intensities and improved system stability compared with the reference measurement procedure. In the range of 3% to 14% HbA(1c), intralaboratory CVs were 0.71% to 1.86%. Deviations from IFCC target values were -0.87 to 1.00 relative %. These values fulfill acceptability criteria for HbA(1c) determination set by the IFCC Working Group on HbA(1c) Standardization. CONCLUSIONS: This procedure for the determination of HbA(1c) improves the existing reference measurement procedure.

Tracing prehistoric activities: musculoskeletal stress marker analysis of a Stone-Age population on the island of Gotland in the Baltic sea.

The skeletal remains from the Middle Neolithic (2750-2300 BC) burial ground at Ajvide, Gotland, are analyzed in order to explore musculoskeletal patterns and to attempt to trace general as well as three specific prehistoric activities (archery, harpooning, and kayaking) that are likely to have been performed in this marine setting of fishing, hunting, and gathering. Scoring of muscular and ligament attachments is performed using the scoring method of Hawkey and Merbs ([1995] Int. J. Osteoarchaeol. 5:324-338) for muskuloskeletal stress markers (MSM). The skeletal material consists of 24 male and 15 female adult individuals divided into three age groups: young (<24 years), middle (25-39 years), and old (>40 years). Thirty upper body MSM sites, on both the left and right sides, are scored and form the basis of the study. Results show that males most frequently have higher mean MSM scores than females. Bilateral asymmetry was noted as low in both sexes. Age proved to be a contributing factor to increased MSM scores, with a greater age-related increase in females. MSM patterns were analyzed statistically in muscle groups associated with the three investigated activities. Significant positive correlations were observed in male individuals in muscle groups associated with archery and to some extent harpooning, an indication that these activities would mainly have been performed by men. Correlations in kayaking muscles were not evidently consistent with the kayaking motion. Furthermore, the costoclavicular ligament, often referred to in connection with "kayaker's clavicle," showed no positive statistical correlation with the kayaking muscles.