Induction of a Regulatory Phenotype in CD3+ CD4+ HLA-DR+ T Cells after Allogeneic Mixed Lymphocyte Culture; Indications of Both Contact-Dependent and -Independent Activation.

Although the observation of major histocompatibility complex II (MHCII) receptors on T cells is longstanding, the explanation for this occurrence remains enigmatic. Reports of an inducible, endogenous expression exist, as do studies demonstrating a protein acquisition from other cells by mechanisms including vesicle transfer. Irrespective of origin, the presence of the human MHCII isotype, human leukocyte antigen DR (HLA-DR), potentially identifies a regulatory T cell population. Using an allogeneic mixed lymphocyte culture (MLC) to induce an antigen-specific immune response, the role of antigen-presenting cells (APCs) for the presence of HLA-DR on cluster of differentiation 3(CD3)+ CD4+ T cells was evaluated. Moreover, a functional phenotype was established for these T cells. It was demonstrated that APCs were essential for HLA-DR on CD3+ CD4+ T cells. Additionally, a regulatory T cell phenotype was induced in CD3+ CD4+ HLA-DR+ responder T cells with an expression of CD25, CTLA-4, CD62L, PD-1, and TNFRII. This phenotype was induced both with and without physical T cell:APC contact, which could reveal novel indications about its functionality. To further investigate contact-independent communication, a phenotype of the small cell-derived vesicles from the MLCs was determined. Yet heterogeneous, this vesicle phenotype displayed contact-dependent differences, providing clues about their intended function in cellular communication.

Simultaneous occurrence of foetal and neonatal alloimmune thrombocytopenia and neonatal neutropenia due to maternal neutrophilic autoantibodies: a case study and review of the literature.

UNLABELLED: Foetal and neonatal alloimmune thrombocytopenia (FNAIT) and neonatal neutropenia caused by maternal autoantibodies against neutrophils are rare disorders. We describe a newborn with severe thrombocytopenia and intracerebral bleeding caused by maternal anti-HPA-3a alloantibodies and mild neutropenia caused by maternal autoantibodies against HNA-1b. This appears to be the first case of simultaneous occurrence of these two conditions. CONCLUSION: This case report and review of the literature demonstrate that anti-HPA-3a antibodies can be overlooked by standard assays.

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.

Bone marrow aspiration technique may have an impact on therapy stratification in children with acute lymphoblastic leukaemia.

BACKGROUND: Morphological evaluation of early response to chemotherapy and measurement of minimal residual disease by flow cytometry or PCR are being used for evaluation of prognosis and treatment stratification in children with acute lymphoblastic leukaemia (ALL). PROCEDURE: In a series of 14 consecutive bone marrow investigations from children with precursor B-cell ALL, morphological evaluations of smears and flow cytometric measurements of minimal residual disease in sequentially aspirated small (2 ml) and large (5-10 ml) volumes of bone marrow were compared, at various time points during therapy. RESULTS: The density of nucleated cells was markedly reduced in the large volume aspirate. The percentage of erythroblasts measured by flow cytometry was smaller, indicating dilution with peripheral cells. Similarly, the blast percentage was reduced with 54% in large aspirates, and in four instances with minimal residual disease of >0.1% in the small volume, the level of blasts in the large aspirate was below this limit. CONCLUSIONS: The amount of minimal residual disease should be measured in the first 2.5 ml of bone marrow aspirated from one puncture site. The procedure should be performed by experienced and carefully instructed doctors. In large aspirates, minimal residual disease will be underestimated, which may lead to failure to undertake a required intensification of therapy and a lower fraction of high risk patients in the trial.

Treatment with intravenous immunoglobulin increases the level of small EVs in plasma of pregnant women with recurrent pregnancy loss.

Extracellular vesicles (EVs), which are small cell-derived compartments, take part in numerous different physiological processes. The contents of EVs reveal the cell of origin and indicates pathophysiological states in different diseases. In pregnancy disorders, changes have been reported in the composition, bioactivity and concentration of placental and non-placental EVs. The purpose of this study was to monitor the effects on EVs in patients receiving intravenous immunoglobulin (IVIG) or placebo (albumin) treatment due to recurrent pregnancy loss (RPL). In a placebo-controlled trial study of IVIG treatment, plasma collected from 39 women with RPL were investigated using the Extracellular Vesicle Array (EV Array). Plasma was sampled consecutively (from gestational week (GW) 5) and the protein phenotypes of the smaller EVs (sEVs) were analyzed for the presence of 34 markers. The levels of sEVs or changes in their levels in early pregnancy were correlated with treatment. There was statistically significant increased levels of sEVs in patients who received IVIG versus placebo. In conclusion, the treatment with high-doses of IVIG clearly boosted the production and release of sEVs to the circulation; however, the biological role of this boost remains to be clarified in further studies.

Mannose-binding lectin-2 genotypes and recurrent late pregnancy losses.

BACKGROUND: Low levels of mannose-binding lectin (MBL) predispose to various infectious and inflammatory disorders and have been reported to be associated with recurrent early miscarriages. Recurrent late pregnancy losses (RLPL) in the second trimester is a rare but devastating syndrome where maternal rather than fetal causes are likely to play a stronger role than in early recurrent miscarriage. METHODS: We identified 75 patients with at least two late losses of pregnancies with apparently normal fetuses between gestational week 14 and 30 among patients with recurrent pregnancy losses referred to our clinic. Polymorphisms in the MBL2 gene associated with plasma MBL levels were investigated in all patients and in 104 women with two or more children and no miscarriages. The patients were divided into three groups: one with clinical signs of cervical insufficiency, one positive for the lupus anticoagulant (LAC) and an idiopathic group. RESULTS: Among all patients with RLPL, 26.7% had MBL2 genotypes associated with MBL deficiency compared with 12.5% in controls [odds ratio (OR) 2.55; 95% confidence interval (CI) 1.17-5.52; P < 0.02]. Among patients with clinical signs of cervical insufficiency or the LAC, the frequency of genotypes associated with MBL deficiency was not significantly increased. However, among 38 patients with idiopathic RLPL, 36.8% carried low-producing MBL2 genotypes, which was significantly more than in controls (OR 4.08, 95% CI 1.70-9.83, P = 0.001). CONCLUSIONS: MBL deficiency is strongly associated with idiopathic RLPL. This may point towards a role for excessive inflammatory disturbances as a cause of the syndrome.

[Myalgic encephalomyelitis or chronic fatigue syndrome].

In this review, we discuss the myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), which is characterised by extreme mental and physical fatigue with associated symptoms of pain, disturbed sleep, cognitive and autonomic dysfunction, as well as post-exertional malaise. This con-dition is often preceded by an infection, severe physiological and/or psychological strain. Over the last decades, research has demonstrated mitochondrial, neuroendocrine, immuno-logical, and metabolic perturbations in patients with ME/CFS, giving hope for the development of new biomarkers and new treatment modalities.

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.

Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen.

Anti-Rhesus D immunoglobulin purified from human sera is used as a prophylactic reagent in Rhesus D negative women at risk of alloimmunization during pregnancy. We are currently developing a Rhesus D antigen-specific recombinant polyclonal antibody drug lead for replacing the existing blood derived-products. By analyzing the RhD-specific antibody VH repertoires from eight alloimmunized women we found, in agreement with previous studies, a strong preference for the VH 3-33 "superspecies" gene segments which encompasses the IGHV3-30-3*01, IGHV3-30*18, and IGHV3-33*01 VH alleles. Even more extensive genetic restriction was observed among five donors, which produced antibodies of identical V-D-J usage and CDR3 loop length and joining regions of similar amino acid composition. In addition, we find a high degree of sequence relatedness to previously isolated anti-Rhesus D antibodies. Such close homology in VH domains indicates that significant structural restrictions are operating in the selection of antibodies recognizing RhD as seen for T cell receptors. Moreover, some VH domains were isolated in their germline configuration indicating that anti-RhD antibodies of relatively high affinity are present in the naïve antibody repertoire of Rhesus negative individuals which offers an explanation for the strong and clinically significant immunogenicity of the Rhesus D.

The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray.

The research field of extracellular vesicles (EVs) is increasing immensely and the potential uses of EVs seem endless. They are found in large numbers in various body fluids, and blood samples may well serve as liquid biopsies. However, these small membrane-derived entities of cellular origin are not straightforward to work with in regard to isolation and characterization. A broad range of relevant preanalytical issues was tested, with a focus on the phenotypic impact of smaller EVs. The influences of the i) blood collection tube used, ii) incubation time before the initial centrifugation, iii) transportation/physical stress, iv) storage temperature and time (short term and long term), v) choice of centrifugation protocol, vi) freeze-thaw cycles, and vii) exosome isolation procedure (ExoQuick™) were examined. To identify the impact of the preanalytical treatments, the relative amounts (detected signal intensities of CD9-, CD63- and/or CD81-positive) and phenotypes of small EVs were analyzed using the multiplexed antibody-based microarray technology, termed the EV Array. The analysis encompassed 15 surface- or surface-related markers, including CD9, CD63, CD81, CD142, and Annexin V. This study revealed that samples collected in different blood collection tubes suffered to varying degrees from the preanalytical treatments tested here. There is no unequivocal answer to the questions asked. However, in general, the period of time and prospective transportation before the initial centrifugation, choice of centrifugation protocol, and storage temperature were observed to have major impacts on the samples. On the contrary, long-term storage and freeze-thawing seemed to not have a critical influence. Hence, there are pros and cons of any choice regarding sample collection and preparation and may very well be analysis dependent. However, to compare samples and results, it is important to ensure that all samples are of the same type and have been handled similarly.

Characterization of a Cell-Culturing System for the Study of Contact-Independent Extracellular Vesicle Communication.

Appropriate and well-documented in vitro cell-culturing systems are necessary to study the activity and biological function of extracellular vesicles (EVs). The aim of this study was to describe an experimental system, in which dynamic, vesicle-based cell communication can be investigated. A commercially available cell-culturing system was applied to study contact-independent cell communication, which separated two cell populations using a membrane with a pore size of 0.4 µm. The EV exchange characteristics between the two compartments in the culture set-up was preliminarily investigated in a cell-free set-up, and analysed using the Extracellular Vesicle (EV) Array and Nanoparticle Tracking Analysis. The application of the cell-culturing set-up was demonstrated using co-cultures of human primary cells. The effects of the relative placement of the two cell populations on the phenotype of EVs found in the cell supernatant were investigated. The results indicate that this placement can be important for the biological hypothesis that is being investigated. These observations are relevant for short (<24h) as well as long (several days) studies of vesicle-based cell communication. Moreover, the introduced cell-culturing set-up and analytical strategy can be used to study contact-independent vesicle communication in a reproducible manner.

Phenotyping of Leukocytes and Leukocyte-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

Exosomal Proteins as Diagnostic Biomarkers in Lung Cancer.

INTRODUCTION: Exosomes have been suggested as promising biomarkers in NSCLC because they contain proteins from their originating cells and are readily available in plasma. In this study, we explored the potential of exosome protein profiling in diagnosing lung cancers of all stages and various histological subtypes in patients. METHODS: Plasma was isolated from 581 patients (431 with lung cancer and 150 controls). The extracellular vesicle array was used to phenotype exosomes. The extracellular vesicle array contained 49 antibodies for capturing exosomes. Subsequently, a cocktail of biotin-conjugated CD9, CD81, and CD63 antibodies was used to detect and visualize captured exosomes. Multimarker models were made by combining two or more markers. The optimal multimarker model was evaluated by area under the curve (AUC) and random forests analysis. RESULTS: The markers CD151, CD171, and tetraspanin 8 were the strongest separators of patients with cancer of all histological subtypes versus patients without cancer (CD151: AUC = 0.68, p = 0.0002; CD171: AUC = 0.60, p = 0.0002; and TSPAN8: AUC = 0.60, p = 0.0002). The multimarker models with the largest AUC in the cohort of patients with all lung cancer histological subtypes and in the cohort of patients with adenocarcinoma only covered 10 markers (all cancer: AUC = 0.74 [95% confidence interval: 0.70-0.80]; adenocarcinoma only: AUC = 0.76 [95% confidence interval: 0.70-0.83]). In squamous cell cancer and SCLC, multimarker models did not exceed CD151 as an individual marker in separating patients with cancer from controls. CONCLUSION: We have demonstrated exosome protein profiling to be a promising diagnostic tool in lung cancer independently of stage and histological subtype. Multimarker models could make a fair separation of patients, demonstrating the perspectives of exosome protein profiling as a biomarker.

Oxygen-Related Differences in Cellular and Vesicular Phenotypes Observed for Ovarian Cell Cancer Lines.

Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients compared to healthy individuals, possibly signifying the presence of an aberrant process. The hypoxia-induced release of EVs from cancer cells has been hypothesized to cause the malignant transformation of healthy recipient cells. In this study, the phenotype of cells and EVs from the ovarian cancer cell lines, COV504, SKOV3, and Pt4, were quantified and analysed under normoxic and hypoxic conditions. It was shown that both cells and EVs express common markers and that the EV phenotype varies more than the cellular phenotype. Additionally, cells subjected to 24 hours of hypoxia compared to normoxia produced more EVs. The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.

Potentials and capabilities of the Extracellular Vesicle (EV) Array.

Extracellular vesicles (EVs) and exosomes are difficult to enrich or purify from biofluids, hence quantification and phenotyping of these are tedious and inaccurate. The multiplexed, highly sensitive and high-throughput platform of the EV Array presented by Jørgensen et al., (J Extracell Vesicles, 2013; 2: 10) has been refined regarding the capabilities of the method for characterization and molecular profiling of EV surface markers. Here, we present an extended microarray platform to detect and phenotype plasma-derived EVs (optimized for exosomes) for up to 60 antigens without any enrichment or purification prior to analysis.

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.

Novel insertions of Bruton tyrosine kinase in patients with X-linked agammaglobulinemia.

Mutations in the gene encoding Bruton tyrosine kinase (BTK) result in X-linked agammaglobulinemia (XLA), an immunodeficiency of antibody defect. By using base excision sequence scanning method (BESS) followed by direct sequencing we found in seven unrelated families with a classical XLA phenotype various mutations including six novel mutations (g.64512_64513insC, c.108_109insG, c.1700_1701insACTACAG, g.51375_51376GC>TG, g.63991_63992insGGTAGAAAAAA, c.1956_1957insCA) and a previously known silent polymorphism (c.2031C>T). Except for two mutations, the alterations affect the kinase domain. There was exceptionally high proportion of insertions in the cohort. Frameshift insertion was found altogether in five patients, three of which are on introns, one in upstream region, and one in exon 18 leading to frameshift mutation and truncation of the protein. In the intron 4 there is a substitution of two bases. Carrier detection was performed in four families. In one case the mutation was found to be de novo.

[TRALI is an overlooked severe complication related to blood transfusion].

Transfusion-related acute lung injury (TRALI) is recognized as the most frequent cause of transfusion-related severe morbidity and mortality. TRALI is characterized by post-transfusional respiratory distress, hypoxaemia and radiographic verified lung infiltration, in the absence of sign of circulatory overload. TRALI is predominantly triggered by human leukocyte antigen or human neutrophil antigen (HNA) antibodies from the transfused blood component. Particularly antibodies against the HNA-3a are involved in severe and fatal TRALI cases. The serological investigation is important to trace and exclude blood donors with TRALI antibodies.

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.