Kim Varming

Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping

Background Exosomes are one of the several types of cell-derived vesicles with a diameter of 30–100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Methods Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. Results The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 104 cells was needed to obtain signals or that only 2.5×104 exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1–10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.

Multifactorial Etiology of Recurrent Miscarriage and Its Scientific and Clinical Implications

A considerable proportion of recurrent miscarriage (RM) cases are caused by recurrent chromosomally abnormal conceptions. However, in younger patients and patients with multiple miscarriages, maternal causes seem to dominate. No single biomarker with a high predictive value of maternally caused RM has been identified. Non-genetic biomarkers in RM may not reflect conditions in the pregnant uterus and we rarely know whether they are causes or consequences of miscarriage. Studies of genetic biomarkers are probably the best way to reveal the pathophysiological mechanisms behind RM. Epidemiological and genetic studies suggest that RM due to maternal causes has a multifactorial background. The risk of RM in each patient is probably determined by the interaction of many genetic variants and environmental factors but only few of these have so far been identified. The genetic biomarkers for RM can probably be classified into three groups: (1) variants associated with excessive inflammatory responses and autoimmunity; (2) variants of importance for insulin and androgen sensitivity and turn-over, and (3) variants associated with thrombophilia. Identification of these markers will require whole genome association studies comprising thousands of individuals. Acknowledgement of the multifactorial background for RM has important implications for the management of patients in clinical practice.

Long-term supplementation with n-3 fatty acids, II: Effect on neutrophil and monocyte chemotaxis.

The effect of a daily supplement with 4 g of n-3 polyunsaturated fatty acids (PUFA) for 9 months to 24 healthy volunteers on neutrophil and monocyte chemotaxis was studied using the under-agarose technique. Autologous serum and n-formyl-methionyl-leucyl-phenylalanine were used as chemoattractants. The effect after 9 months of supplementation with n-3 PUFA was also compared to results after short-term supplementation with n-3 PUFA for 6 weeks. Monocyte chemotaxis was reduced after 9 months of supplementation with n-3 PUFA to the same extent as after 6 weeks supplement. Neutrophil-directed migration towards chemoattractants was reduced after 9 months on fish oil, and this decrease was significantly greater than the decrease obtained after 6 weeks of supplementation. The spontaneous migration of neutrophils was significantly attenuated after 9 months compared to baseline and to 6 weeks. These findings lend support to a role for n-3 PUFA in the management of chronic inflammatory and atherosclerotic vascular diseases.

Diagnostic and prognostic potential of extracellular vesicles in peripheral blood

PURPOSE Extracellular vesicles (EVs) are small, membrane-enclosed entities released from cells in many different biological systems. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. The complement of biomolecules reflects the parent cell, and their characterization may provide information about the presence of an aberrant process. Peripheral blood is a rich source of circulating EVs, which are easily accessible through a blood sample. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic and prognostic value. The objectives of this review are to briefly present the current knowledge about EVs and to introduce a toolbox of selected techniques, which can be used to rapidly characterize clinically relevant properties of EVs from peripheral blood. METHODS Several techniques exist to characterize the different features of EVs, including size, enumeration, RNA cargo, and protein phenotype. Each technique has a number of advantages and pitfalls. However, with the techniques presented in this review, a possible platform for EV characterization in a clinical setting is outlined. FINDINGS Although EVs have great diagnostic and prognostic potential, a lack of standardization regarding EV analysis hampers the full use of this potential. Nevertheless, the analysis of EVs in peripheral blood has several advantages compared with traditional analyses of many soluble molecules in blood. IMPLICATIONS Overall, the use of EV analysis as a diagnostic and prognostic tool has prodigious clinical potential.

No effect of a very low dose of n-3 fatty acids on monocyte function in healthy humans

There is some evidence that intake of sea food rich in n-3 polyunsaturated fatty acids (PUFA) may protect against coronary heart disease (CHD). Thus, even very low daily amounts of n-3 PUFA below 0.5 g have been reported to reduce the incidence of CHD, although it is unknown by what mechanisms this may occur. Monocytes are of major importance in atherogenesis, and we therefore studied the effect of a daily supplement with 0.65 g of n-3 PUFA for 12 weeks on monocyte function in a randomized placebo-controlled trial of 32 healthy subjects. No effect of dietary n-3 PUFA could be demonstrated on monocyte chemotaxis, on chemiluminescence, or on formation of cytokines and leukotriene B4 from activated monocytes.

Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma

Background Lung cancer is one of the leading causes of cancer-related death. At the time of diagnosis, more than half of the patients will have disseminated disease and, yet, diagnosing can be challenging. New methods are desired to improve the diagnostic work-up. Exosomes are cell-derived vesicles displaying various proteins on their membrane surfaces. In addition, they are readily available in blood samples where they constitute potential biomarkers of human diseases, such as cancer. Here, we examine the potential of distinguishing non-small cell lung carcinoma (NSCLC) patients from control subjects based on the differential display of exosomal protein markers. Methods Plasma was isolated from 109 NSCLC patients with advanced stage (IIIa–IV) disease and 110 matched control subjects initially suspected of having cancer, but diagnosed to be cancer free. The Extracellular Vesicle Array (EV Array) was used to phenotype exosomes directly from the plasma samples. The array contained 37 antibodies targeting lung cancer-related proteins and was used to capture exosomes, which were visualised with a cocktail of biotin-conjugated CD9, CD63 and CD81 antibodies. Results The EV Array analysis was capable of detecting and phenotyping exosomes in all samples from only 10 µL of unpurified plasma. Multivariate analysis using the Random Forests method produced a combined 30-marker model separating the two patient groups with an area under the curve of 0.83, CI: 0.77–0.90. The 30-marker model has a sensitivity of 0.75 and a specificity of 0.76, and it classifies patients with 75.3% accuracy. Conclusion The EV Array technique is a simple, minimal-invasive tool with potential to identify lung cancer patients.

The effect of n-3 fatty acids on neutrophil chemiluminescence

The effect of dietary supplementation with n-3 polyunsaturated fatty acids (PUFAs) on the production of free oxygen radicals from activated neutrophils was investigated in healthy subjects, using chemiluminescence. In the first study 22 persons were give 4 g n-3 PUFAs daily for 6 weeks. There was a median reduction of chemiluminescence from neutrophils stimulated with opsonized zymosan of 37% (p < 0.001). The median content of eicosapentaenoic acid in platelets, used as an indicator for cellular fatty acid profile, increased from 0.70 to 2.80% (p < 0.001), and there was a significant negative correlation between the chemiluminescence signal and the content of eicosapentaenoic acid in platelets (p < 0.001). In a second, low-dose study 24 persons were allocated to daily supplementation with either 0.65 g n-3 PUFAs or with a control oil for 6 weeks. Compared to the control group there was a median reduction of 38 and 44% in chemiluminescence from neutrophils stimulated with opsonized zymosan and phorbol,12-myristate,13-acetate (PMA), respectively. Neither of these differences, however, was statistically significant. These findings lend support for a possible role of n-3 PUFAs in the management of inflammatory disorders.

n-3 fatty acids and leukocyte chemotaxis. Effects in hyperlipidemia and dose-response studies in healthy men.

Dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFAs) has been shown to inhibit neutrophil and monocyte chemotaxis in healthy subjects and, with respect to neutrophils, also in various patient groups. We studied the effect of dietary supplementation with n-3 PUFAs on monocyte and neutrophil chemotaxis in patients with hyperlipidemia. Chemotaxis was investigated with the under-agarose assay, using autologous serum and N-formyl-methionyl-leucyl-phenylalanine as chemoattractants. The patients were examined before and after 6 weeks of supplementation with 6 g n-3 PUFAs daily. Monocyte chemotaxis was reduced after n-3 PUFA supplementation in type IIa patients but was unaffected in patients with type IV hyperlipidemia. Furthermore, monocyte chemotaxis was increased in untreated type IIa patients compared with normocholesterolemic controls. We also studied the dose-response effects of n-3 PUFAs on monocyte and neutrophil chemotaxis in healthy men given 1.3, 4, and 9 g n-3 PUFAs daily for 6-week periods. Monocyte and neutrophil chemotaxis was reduced after n-3 PUFA supplements in a dose-dependent fashion, with the majority of the effect observed after the low dose. These results lend support to the notion of an antiatherosclerotic effect of n-3 PUFAs and may provide an explanation for the hitherto-unexplained effect of low doses of n-3 PUFAs in coronary heart disease.

FcγRIIIB polymorphism: evidence that NA1/NA2 and SH are located in two closely linked loci and that the SH allele is linked to the NA1 allele in the Danish population

BACKGROUND: The neutrophil‐specific antigens NA1, NA2, and SH are well‐recognized allotypic forms of FcγRIIIB. Individuals carrying all three FcγRIIIB genes were recently described.

Association of HY-restricting HLA class II alleles with pregnancy outcome in patients with recurrent miscarriage subsequent to a firstborn boy

Healthy females, pregnant with a boy, generate immune responses against male-specific minor histocompatibility (HY) antigens. The clinical importance of these responses is evident in stem cell transplantation. Birth of a boy prior to a series of miscarriages reduces the chance of a subsequent live birth. This study explores the putative impact of known HY-presenting HLA alleles on future pregnancy outcome in women with at least three consecutive miscarriages following a birth [secondary recurrent miscarriage (SRM)]. HLA-A, -B, -DRB1, DRB3-5 and DQB1 genotyping was performed in 358 SRM patients and in 203 of their children born prior to the miscarriages. The subsequent live birth in women with boys prior to the miscarriages compared with girls is lower in women with HY-restricting HLA class II alleles [odds ratio (OR): 0.17 (0.1-0.4), P = 0.0001]. One HY-restricting HLA class II allele in women with firstborn boys significantly reduces the chances of a live birth [OR: 0.46 (0.2-0.9), P = 0.02]. Two HY-restricting HLA class II alleles further reduced this chance [OR: 0.21 (0.1-0.7), P = 0.02]. HY-restricting HLA class II did not reduce the chances of a live birth in SRM women with firstborn girls. HY-restricting HLA class II alleles are associated with a decreased chance of a live birth in SRM women with firstborn boys. These findings strongly indicate an aberrant maternal immune reaction against fetal HY-antigens in SRM. The results may shed light on the as-yet unknown immunological causes of SRM and may help understand the successful maternal acceptance of the fetal semi-allograft.