Presence of HLA-DR Molecules and HLA-DRB1 mRNA in Circulating CD4(+) T Cells.

The human major histocompatibility complex class II isotype HLA-DR is currently used as an activation marker for T cells. However, whether an endogenous protein expression or a molecular acquisition accounts for the presence of HLA-DR on T cells remains undetermined and still controversial. To further characterize this phenomenon, we compared several aspects of the presence of the HLA-DR protein to the presence of associated mRNA (HLA-DRB1), focusing on human T cells from peripheral blood of healthy individuals. Using a flow cytometric approach, we determined that the HLA-DR observed on CD4(+) T cells was almost exclusively cell surface-associated, while for autologous CD19(+) B cells, the protein could be located in the plasma membrane as well as in the cytoplasm. Moreover, negligible expression levels of HLA-DRB1 were found in CD4(+) T cells, using an HLA-DRB1 allele-specific qPCR assay. Finally, the presence of HLA-DR was not confined to activated CD4(+) and CD8(+) T cells, as evaluated by the co-expression of CD25. The functional role of the HLA-DR molecule on T cells remains enigmatic; however, this study presents evidence of fundamental differences for the presence of HLA-DR on T cells from HLA-DR in the context of antigen-presenting cells, which is a well-known phenomenon. Although an inducible endogenous protein expression cannot be excluded for the T cells, our findings suggest that a re-evaluation of the HLA-DR as a T cells activation marker is warranted.

Does smoking, age or gender affect the protein phenotype of extracellular vesicles in plasma?

Extracellular vesicles (EVs) are involved in several diseases, which have formed the basis for the potential use of EV analyses in a clinical setting. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. With this extensive study of 161 healthy individuals it was elucidated that certain markers of plasma EVs are influenced by demographic variations such as gender, age and smoking status. When the purpose is to use EVs as a diagnostic tool, it should be emphasized how important it is to choose the correct demographic group when comparing marker levels of plasma EVs.

In vitro Assay for Screening of Optimal Targets for Antigen-Delivery to Murine Dendritic Cells.

Targeting of antigen to dendritic cells (DCs) increase the efficiency of immunization procedures and may facilitate the development of more effective vaccines. Several surface molecules on DCs have shown to be useful for antigen targeting, but many more deserves investigation for their efficacy in this respect. With this end in mind, a simple in vitro assay for screening of optimal targets for antigen-delivery to murine DCs was established. Splenocytes from mice immunized with rat IgG were targeted in vitro with a panel of different rat monoclonal antibodies (mAbs) directed against surface markers on murine DCs. The resulting T-cell activation was analysed by determining the number of IFN-γ and IL-4 secreting cells by ELISPOT. A positive effect of targeting was evident with several of the mAbs. Thus, mAbs against CD11c, CD36, CD205 and Clec7A all induced IFN-γ responses that were significantly higher than those induced by non-targeting control mAbs. Anti-CD36 also induced IL-4 responses that were significantly higher than the control. The assay described here allows simultaneous analysis of a large number of potential target structures and facilitates direct comparison between the different targets regarding the strength of the T-cell responses induced by the targeted DCs. The assay could be useful as a first-line screening of potential target structures on murine DCs.

Enhanced Humoral Responses Induced by Targeting of Antigen to Murine Dendritic Cells.

Dendritic cells (DCs) are superior in their ability to induce and control adaptive immune responses. These qualities have motivated the hypothesis that targeted delivery of antigen to DCs in vivo may be an effective way of enhancing immunization. Recent results show that antigen targeted to certain DC surface molecules may indeed induce robust immune responses. Targeting of antigen to DCs can be accomplished by the means of monoclonal antibodies. This study compared the humoral responses induced in mice by in vivo targeting of DCs using monoclonal antibodies specific for CD11c, CD36, CD205, Clec6A, Clec7A, Clec9A, Siglec-H and PDC-TREM. The results demonstrate that antigen delivery to different targets on DCs in vivo gives rise to humoral responses that differ in strength. Targeting of antigen to CD11c, CD36, CD205, Clec6A, Clec7A and PDC-TREM induced significantly stronger antibody responses compared to non-targeted isotype-matched controls. Targeting of Clec9A and Siglec-H did not lead to efficient antibody responses, which may be due to unfavourable properties of the targeting antibody, in which case, other antibodies with the same specificity might elicit a different outcome. Anti-CD11c was additionally used for elucidating the impact of the route of vaccination, and the results showed only minor differences between the antibody responses induced after immunization either s.c., i.v. or i.p. Altogether, these data show that targeting of different surface molecules on DCs result in very different antibody responses and that, even in the absence of adjuvants, strong humoral responses was induced.

Frequencies of HNA-1, HNA-3, HNA-4, and HNA-5 in the Danish and Zambian populations determined using a novel TaqMan real time polymerase chain reaction method.

In this study, we report a novel real time polymerase chain reaction (Q-PCR) method using TaqMan probes for human neutrophil antigens (HNA)-1, -3, -4, and -5 genotyping. The method was validated in a Caucasian Danish population, a Zambian population, and in clinical samples using three different methods: an in-house polymerase chain reaction with sequence-specific primers (PCR-SSP) method, a commercial available PCR-SSP kit and a novel Q-PCR method. We observed no discrepancy in the genotype frequencies determined by the PCR-SSP methods and the TaqMan assay in the populations studied. In tests of a family of Nigerian origin and in samples carrying the rare SLC44A2*1:2 genotype, different results were produced by the commercial PCR-SSP kit and the real-time TaqMan assay. The TaqMan-based genotyping method was rapid and reproducible, allowing high-throughput HNA-1, -3, -4, and -5 genotyping.

Early and late markers for the detection of early-onset neonatal sepsis.

INTRODUCTION: In this study we tested how a combination of early and late paraclinic markers could predict early onset neonatal sepsis (EONS). METHODOLOGY: The first 24 hours after the suspicion of EONS, we measured interleukine (IL)-6, IL-8, IL-10, IL-18, tumor necrosis factor-alpha (TNF-alpha), interferon gamma (INF-gamma), procalcitonin (PCT) and C-reactive protein (CRP) at 8-hour intervals on 123 neonates clinically suspected for EONS. The neonates were divided into two groups. The sepsis group: 1A with blood culture verified bacteraemia and 1B strongly suspected sepsis (29 patients). The no sepsis group: 2A treated with antibiotics (37 patients) and 2B not treated with antibiotics (57 patients). RESULTS: Combined evaluation of each of the early markers with PCT > 25 ng/ml for prediction of EONS at time 0, gave the following sensitivities and specificities: IL-6 > 250 pg/ml: 71% and 88%; IL-8 > 900 pg/ml: 50% and 88%; IL-10 > 40 pg/ml: 43% and 87%; and immature/total (I/T) ratio > 0.35: 59% and 88%. The results of IL-18, TNF-alpha and IFN-gamma did not predict EONS. CONCLUSION: IL-6 combined with PCT values is a fair way to evaluate EONS at the time of suspicion of infection. The "old" early marker, I/T ratio, is almost as efficient as IL-6. By combining an early and a late marker it may be possible to reduce the diagnostic "non-conclusive" period of paraclinic values.

Exosomal proteins as prognostic biomarkers in non-small cell lung cancer.

BACKGROUND: Use of exosomes as biomarkers in non-small cell lung cancer (NSCLC) is an intriguing approach in the liquid-biopsy era. Exosomes are nano-sized vesicles with membrane-bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection for optimal treatment. We here evaluate exosomes by protein phenotyping as a prognostic biomarker in NSCLC. METHODS: Exosomes from plasma of 276 NSCLC patients were phenotyped using the Extracellular Vesicle Array; 49 antibodies captured the proteins on the exosomes, and a cocktail of biotin-conjugated antibodies binding the general exosome markers CD9, CD81 and CD63 was used to visualise the captured exosomes. For each individual membrane-bound protein, results were analysed based on presence, in a concentration-dependent manner, and correlated to overall survival (OS). RESULTS: The 49 proteins attached to the exosomal membrane were evaluated. NY-ESO-1, EGFR, PLAP, EpCam and Alix had a significant concentration-dependent impact on inferior OS. Due to multiple testing, NY-ESO-1 was the only marker that maintained a significant impact on inferior survival (hazard rate (HR) 1.78 95% (1.78-2.44); p = 0.0001) after Bonferroni correction. Results were adjusted for clinico-pathological characteristics, stage, histology, age, sex and performance status. CONCLUSION: We illustrate the promising aspects associated with the use of exosomal membrane-bound proteins as a biomarker and demonstrate that they are a strong prognostic biomarker in NSCLC.

Rapid genotyping of MBL2 gene mutations using real-time PCR with fluorescent hybridisation probes.

In this study, we describe a real-time polymerase chain reaction (PCR) for genotyping all known polymorphisms of the human mannose-binding lectin 2 (MBL2) gene. These comprised two variations in the 5' regulatory region at positions -550 (H/L) and -221 (X/Y), one in the 5' untranslated sequence at position +4 (P/Q) and three structural mutations within exon 1 at codons 52, 54, and 57, also known as the D, B and C variants, respectively. Three reactions with two different conditions were sufficient to genotype one individual unambiguously. The three mutations in exon 1 were detected in one capillary using a sensor probe covering the three mutations, whereas amplification of the variants located upstream of the coding sequence was performed in only two reactions. Single colour detection was used for detection of the (H/L) polymorphism and multiplexing by dual colour probes was used for simultaneous genotyping of (X/Y) and (P/Q). The reliability of the system was evaluated by comparison with a conventional PCR method with sequence-specific primers (PCR-SSP). For this study, 100 individuals of Danish and 30 of African descent were analysed, and the genotypes obtained were concordant in all cases. This new method is rapid and provides reliable results without ambiguities.

Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers.

This study describes a new approach to the determination of all known mannan-binding lectin (MBL) mutations. The distribution of known variants of the MBL gene in a population of healthy unrelated Danes was determined and the genotype was correlated with the plasma MBL concentrations. The following genetic polymorphisms were studied: three point mutations in the promoter region at position -550 (H/L variants), -221 (X/Y variants), -70 (nt C or T), one point mutation in the 5' untranslated (UT) region at position +4 (P/Q variants) and three point mutations located at codons 52, 54 and 57 in exon 1 of the MBL gene, at nucleotide positions 223, 230 and 239, respectively. To perform genotyping, we designed sequence specific primers for a polymerase chain reaction (PCR-SSP). PCR-SSP is a powerful technique for the discrimination of alleles resulting from single base substitutions and is a widely used technique. Another major advantage of the PCR-SSP method is its ability to determine whether sequence motifs are in cis or trans. The frequencies of variants in exon 1 obtained by PCR-SSP were completely comparable to results obtained by previously described PCR methods, restriction fragment length polymorphism (RFLP) and site-directed mutagenesis (SDM). This PCR-SSP method is performed with standard laboratory equipment and has the capacity to detect all genetic variants in 100 samples in 2 days at an estimated total cost of GBP 11 per sample. Analysing the correlation between MBL haplotype and plasma MBL levels, we confirmed that three different structural variants, B, C and D and the promoter haplotypes HY, LY and LX have a dominant effect on the concentration of MBL. The HY haplotype is associated with the highest plasma concentration, the LY haplotype with intermediate levels and the LX haplotype with the lowest levels. The LX haplotype was found to be associated with very low levels of MBL similar to those found in association with the structural B genotype. The gene frequencies of variants in the MBL gene in the Danish population studied correspond to previous reports on Caucasian populations.

Dose-response studies on the effect of n-3 polyunsaturated fatty acids on lipids and haemostasis.

We have studied the dose-response effects of dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA's) on lipids and haemostasis. Ten healthy males were each given 1.3 g, 4 g or 9 g of n-3 PUFA's daily for 6-week periods. Bleeding time, HDL-cholesterol and plasminogen activator inhibitor increased with the dose of n-3 PUFA. Plasma fibrinogen and triglyceride levels were reduced in a dose-dependent fashion. After ingestion of 1.3 g of n-3 PUFA's plasma fibrinogen decreased from 9 to 7 mumol/l and HDL-cholesterol increased from 1.2 to 1.3 mmol/l. The bleeding time was prolonged from 5 to 6.5 min while triglyceride levels decreased from 1.2 to 0.9 mmol/l after ingestion of 4 g of n-3 PUFA's. Dietary supplementation with the highest daily dose (9 g) reduced plasma levels of triglycerides, fibrinogen and von Willebrand factor, while bleeding time, plasminogen activator antigen, plasminogen activator inhibitor and the ratio of HDL-cholesterol to total cholesterol increased.

The effect of n-3 fatty acids on lipids and haemostasis in patients with type IIa and type IV hyperlipidaemia.

Plasma lipids and haemostasis were investigated in 17 patients with hyperlipidaemia before and after 6 weeks supplementation with 6 g n-3 fatty acids. Nine of the patients had type IIa and 8 had type IV hyperlipidaemia. No effect on plasma cholesterol, LDL- or HDL-cholesterol were seen, but plasma triglycerides decreased after n-3 supplementation. Apolipoprotein B increased and apolipoprotein A1 decreased after the oil supplement. The bleeding time was prolonged, but platelet aggregation was unaltered by n-3 fatty acids. Protein C activity increased in type IIa and decreased in type IV after the supplement. Fibrinolysis was markedly depressed while von Willebrand factor antigen was reduced after intake of n-3 fatty acids.

Proinflammatory activation of neutrophils and monocytes by Helicobacter pylori is not associated with cagA, vacA or picB genotypes.

Chronic Helicobacter pylori infection is associated with mucosal inflammation. The aim of the present study was to assess human neutrophil and monocyte activation induced by H. pylori strains with different virulence genotypes. Bacterial sonicates from 12 strains were used to induce phagocyte up-regulation of adherence molecule CD11b, assessed by fluorescence flow cytometry, and oxidative burst responses, assessed by chemiluminescence. A dose-dependent induction of the expression of CD11b was observed with sonicate from all H. pylori strains on both neutrophils and monocytes. Strains negative for cagA and picB genes had the same inducing activity of upregulation of CD11b as strains positive for these genes. A vacA-S2 type strain had the same activity as vacA-S1 type strains. The induction of toxic oxygen radicals by H. pylori-activated neutrophils gave higher median values for the cagA-positive strains than for the cagA-negative strains. For the monocyte chemiluminescence response, cagA-negative strains gave higher median values compared to cagA-positive strains. We conclude that upregulation of the neutrophil and monocyte adherence molecule CD11b induced by H. pylori sonicates is not associated with the presence of cagA, picB or mosaic pattern of vacA, and that cagA, picB-negative strains and vacA-S2 strains retain their inflammatory capacity.

EBV-positive primary central nervous system lymphomas in monozygote twins with common variable immunodeficiency and suspected multiple sclerosis.

Common variable immunodeficiency represents the most frequently occurring primary immunodeficiency disorder and is usually detected sporadically in patients with no family history of immunodeficiency. We present the case stories of two monozygote twins, who following a period of decreasing serum immunoglobulins developed primary central nervous system lymphomas. One twin had clinical and paraclinical features mimicking multiple sclerosis. Immunohistochemical investigations on biopsy tissue showed expression of the bcl-2 and p53 gene products, and Epstein-Barr virus (EBV) encoded small RNA's (EBER) indicating latent infection were detected in lymphoma cells using in situ hybridisation techniques. The pathogenetic role of EBV in oncogenesis is discussed.

The acute effect of a single very high dose of N-3 fatty acids on coagulation and fibrinolysis.

The objective of the study was to investigate the acute effect of a single very high dose of n-3 PUFA on coagulation and fibrinolysis. Forty healthy volunteers were randomized into two groups to receive either 20 grams of n-3 PUFA or 20 grams of n-6 PUFA as a single dose at 6 p.m. with their evening meal. Coagulation and fibrinolysis were evaluated in the fasting state at 8 a.m. the next morning and compared to values obtained at 8 a.m. the day before, when the participants were on their habitual diets. PAI-1 activity in plasma increased by a mean of 62% in subjects randomized to receive n-3 PUFA despite that no changes could be demonstrated in t-PA antigen levels. PAI-1 activity was unaltered in the 20 controls receiving n-6 PUFA. Plasma fibrinogen, coagulation factor VII, thrombin-antithrombin complexes and D-dimer did not significantly change after either supplement. The substantial increase in levels of PAI-1 activity in plasma after a single very high dose of n-3 PUFA may limit the usefulness of single very high doses of n-3 PUFA in acute clinical conditions.

The acute effects of a single very high dose of n-3 fatty acids on plasma lipids and lipoproteins in healthy subjects.

Forty healthy volunteers were allocated in a double blind, randomized study to receive either 20 g of n-3 polyunsaturated fatty acids (PUFA) or 20 g of n-6 PUFA at their evening meal. The effect on plasma lipids and lipoproteins of this single dose of fish oil vs. corn oil was studied the next morning, 14 h after ingestion. Plasma triglycerides and very low density lipoprotein-cholesterol significantly decreased (33%) after n-3 PUFA (P < 0.001), and significantly (P < 0.01) more than after intake of n-6 PUFA. The decrease in plasma triglycerides after n-3 PUFA ingestion was more pronounced in subjects with higher baseline levels of triglycerides (P < 0.001). Total cholesterol decreased after both supplements, but did not differ between the supplements. Low density lipoprotein-cholesterol did not change, and high density lipoprotein-cholesterol significantly decreased in subjects given n-3 PUFA compared to baseline, but not when compared to subjects receiving n-6 PUFA. In conclusion, we have shown that a single very high dose of n-3 PUFA has a pronounced hypotriglyceridemic effect, which is directly related to the initial plasma level.

Determination of alpha-synuclein concentration in human plasma using ELISA.

The nerve cell protein alpha-synuclein is important in Parkinson's disease and dementia with Lewy bodies, and its expression levels are directly linked to development of the diseases. Quantification of the plasma level of alpha-synuclein may therefore be important as a biomarker for disease susceptibility. We present a quantitative measurement of alpha-synuclein in the plasma of healthy control subjects in relation to their age using a novel enzyme-linked immunosorbent assay (ELISA). The plasma concentration among the 44 blood donors displayed a median of 5.6 microg/L (range 2.1-19.4 microg/L) with a narrow distribution (25 % and 75 % percentiles, 4.0 and 7.2 microg/L) and there was no correlation with age and gender. This narrow concentration range and the ease of measuring the quantitative ELISA support future investigations of plasma alpha-synuclein in relation to neurodegenerative diseases.

Novel mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infections.

In 1981 we presented a patient with Mycobacterium intracellulare osteomyelitis and depressed monocyte cytotoxicity. It is now demonstrated that the molecular defect was a never-before-described nucleotide deletion at position 794 (794delT) in the interferon-gamma-receptor alpha-1 gene. The genetic defect was passed on to his daughter who was diagnosed with non-tuberculous mycobacterial osteomyelitis at the age of 7 years.

Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers.

Hereditary haemochromatosis (HH), a condition of abnormal iron metabolism which leads to iron overload and organ damage, previously known as bronze diabetes or idiopathic haemochromatosis, is the most common disease-producing genetic disorder among Europeans. Two mutations, C282Y and H63D, are described for the candidate gene, HFE, reported as being responsible for the disease. Since molecular testing of these mutations will be of value in early diagnosis of haemochromatosis, the aim of this study was to develop a simple, fast and inexpensive technique for the determination of the polymorphism in the HFE gene on a large scale. We designed sequence-specific primers for polymerase chain reaction (PCR-SSP) and tested 200 randomly selected healthy Danes and found the result completely comparable to results obtained by a previously described method, PCR-RFLP. The gene frequencies in the Danish population are similar to reported results for the White population, with a frequency of 0.068 for the C282Y mutation and a frequency of 0.128 for the H63D mutation.

n-3 polyunsaturated fatty acid supplementation (Pikasol) in men with moderate and severe hypertriglyceridaemia: a dose-response study.

The effect of n-3 fatty acids in 11 men with hypertriglyceridaemia was investigated. The subjects were given daily supplements with three different doses of n-3 fatty acids for 8-week periods. The supplements given were 2, 4 and 9 g of n-3 fatty acids/day, respectively. Total cholesterol, triglycerides and the ratio of total cholesterol to HDL-cholesterol significantly decreased with all three doses in a dose-dependent way. HDL-cholesterol increased in a dose-dependent fashion, while apolipoproteins A1 and B were unaltered by the supplements apart from a small increase in apolipoprotein A1 after the lowest dose of n-3 fatty acids. The greatest effect was observed after 2 g of n-3 fatty acids/day.

Prospective, serial investigations of in-vitro lymphocyte cytokine production, CD62L expression and proliferative response to microbial antigens in women with recurrent miscarriage.

BACKGROUND: Lymphocytes from pregnant women with unexplained recurrent miscarriage (RM) may be characterized by a T-helper type 1-dominated cytokine production and a higher proliferative response to microbial recall antigens compared with normal pregnant women. METHODS: Serial blood samples were taken from 14 women with RM (at least three previous consecutive miscarriages) during the first 14 weeks of pregnancy, and one blood sample was taken from 15 control women in gestational weeks 7-8. Of the 14 pregnant RM patients, four produced a live birth and 10 miscarried. Lymphocytes were in-vitro-stimulated by mitogens, allogeneic cells and microbial antigens, and the production of a series of cytokines, the proliferative responses and lymphocytic expression of CD62L (which may be a marker of T-helper type 2 lymphocytes) were measured. RESULTS: Repeated measurements of cytokine production were reproducible during the first trimester. The proliferative responses to herpes simplex and tetanus antigens were increased, and the ratio of CD62L-/CD62L+ expressing CD4+CD45RO+ lymphocytes was decreased in patients compared with controls (P = 0.01, P < 0.01 and P < 0.01 respectively). CONCLUSION: The results of the in-vitro assays used were reproducible in serial testing during pregnancy. The importance of CD62L expression on lymphocytes for RM and the relevance of the maternal response to microbial antigens during pregnancy should be further explored.