Development of the Leiden Independence Questionnaire for Support Staff: a measure of staff behaviour regarding promoting independence of people with intellectual disabilities.

BACKGROUND: Support staff of adults with intellectual disability (ID) play an important role in promoting independence in home and community settings. However, little is known about the types of behaviours staff should use to promote independence and instruments that assess such behaviour do not yet exist. The aim of this study was therefore to develop and initially validate a reliable questionnaire that measures the degree to which support staff display behaviours that promote independence in people with ID. METHOD: The Leiden Independence Questionnaire for Support Staff (LIQSS) was constructed to measure the extent to which support staff promote independence in people with ID. The LIQSS was completed by 142 staff members working with people with ID. For the psychometric evaluation of the LIQSS, a principal component analysis was performed with an oblique rotation in all items. Next, the principal component analysis was performed with a forced three-component extraction, and three sub-scales were computed. To assess internal consistency, Cronbach's α was calculated for each of the sub-scales. RESULTS: The LIQSS was found to consist of three internally consistent (Cronbach's α was respectively 0.92, 0.79 and 0.76) and meaningful components: (1) communication, agreements and coordination; (2) positive encouragement and tailoring; and (3) supporting independent performance. The final 22 items had factor loadings between 0.44 and 0.91 on their corresponding component and a minimal difference in loading to the other factors of 0.20. CONCLUSIONS: The LIQSS appears to be an instrument with positive face validity and reliability (internal consistency) that assesses the degree to which support staff promote independence in people with ID. To increase the instrument's value for both scientific research and clinical practice, studies should focus on the further validation of the LIQSS.

Nanobody-liposomes as novel cancer vaccine platform to efficiently stimulate T cell immunity.

Cancer vaccines can be utilized in combination with checkpoint inhibitors to optimally stimulate the anti-tumor immune response. Uptake of vaccine antigen by antigen presenting cells (APCs) is a prerequisite for T cell priming, but often relies on non-specific mechanisms. Here, we have developed a novel vaccination strategy consisting of cancer antigen-containing liposomes conjugated with CD169- or DC-SIGN-specific nanobodies (single domain antibodies) to achieve specific uptake by APCs. Our studies demonstrate efficient nanobody liposome uptake by human and murine CD169+ and DC-SIGN+ APCs in vitro and in vivo when compared to control liposomes or liposomes with natural ligands for CD169 and DC-SIGN. Uptake of CD169 nanobody liposomes resulted in increased T cell activation by human APCs and stimulated naive T cell priming in mouse models. In conclusion, while nanobody liposomes have previously been utilized to direct drugs to tumors, here we show that nanobody liposomes can be applied as vaccination strategy that can be extended to other receptors on APCs in order to elicit a potent immune response against tumor antigens.

Liposome induction of CD8+ T cell responses depends on CD169+ macrophages and Batf3-dependent dendritic cells and is enhanced by GM3 inclusion.

Cancer vaccines aim to efficiently prime cytotoxic CD8+ T cell responses which can be achieved by vaccine targeting to dendritic cells. CD169+ macrophages have been shown to transfer antigen to dendritic cells and could act as an alternative target for cancer vaccines. Here, we evaluated liposomes containing the CD169/Siglec-1 binding ligand, ganglioside GM3, and the non-binding ligand, ganglioside GM1, for their capacity to target antigens to CD169+ macrophages and to induce immune responses. CD169+ macrophages demonstrated specific uptake of GM3 liposomes in vitro and in vivo that was dependent on a functional CD169 receptor. Robust antigen-specific CD8+ and CD4+ T and B cell responses were observed upon intravenous administration of GM3 liposomes containing the model antigen ovalbumin in the presence of adjuvant. Immunization of B16-OVA tumor bearing mice with all liposomes resulted in delayed tumor growth and improved survival. The absence of CD169+ macrophages, functional CD169 molecules, and cross-presenting Batf3-dependent dendritic cells (cDC1s) significantly impaired CD8+ T cell responses, while B cell responses were less affected. In conclusion, we demonstrate that inclusion of GM3 in liposomes enhance immune responses and that splenic CD169+ macrophages and cDC1s are required for induction of CD8+ T cell immunity after liposomal vaccination.

CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo.

Bone marrow-derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD8(+) T cells in a process referred to as cross-priming. Cross-priming is essential for the induction of CD8(+) T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are capable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded beta2-microglobulin-deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter associated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol transport. To determine whether a specific subset of splenic DCs is involved in this cross-presentation, we negatively and positively selected for CD8(-) and CD8(+) DCs. Only the CD8(+), and not the CD8(-), DC subset demonstrates cross-priming ability. FACS((R)) studies after injection of splenocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD8(+) and the CD8(-) DC subsets, respectively, had one or more associated beads. These results indicate that CD8(+) DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens.

Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein.

H-Y is a transplantation antigen that can lead to rejection of male organ and bone marrow grafts by female recipients, even if the donor and recipient match at the major histocompatibility locus of humans, the HLA (human leukocyte antigen) locus. However, the origin and function of H-Y antigens has eluded researchers for 40 years. One human H-Y antigen presented by HLA-B7 was identified as an 11-residue peptide derived from SMCY, an evolutionarily conserved protein encoded on the Y chromosome. The protein from the homologous gene on the X chromosome, SMCX, differs by two amino acid residues in the same region. The identification of H-Y may aid in transplantation prognosis, prenatal diagnosis, and fertilization strategies.

Identification of a graft versus host disease-associated human minor histocompatibility antigen.

Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.

The minor histocompatibility antigen HA-1: a diallelic gene with a single amino acid polymorphism.

The minor histocompatibility antigen (mHag) HA-1 is the only known mHag for which mismatching is correlated with the development of severe graft versus host disease (GvHD) after human leukocyte antigen-identical bone marrow transplantation. HA-1 was found to be a nonapeptide derived from an allele of the KIAA0223 gene. The HA-1-negative allelic counterpart encoded by KIAA0223 had one amino acid difference from HA-1. Family analysis with HA-1 allele-specific polymerase chain reaction showed an exact correlation between this allelic polymorphism and the HA-1 phenotype. HA-1 allele typing of donor and recipient should improve donor selection and allow the determination of bone marrow transplantation recipients with high risk for HA-1-induced GvHD development.

Early cellular interactions and immune transcriptome profiles in human factor VIII-exposed hemophilia A mice.

Essentials Initial immune cell interactions leading to factor (F) VIII immunity are not well characterized. We assessed cellular interactions and expression profiles in hemophilia A mice. MARCO+, followed by SIGLEC1+ and SIGNR1+ macrophages co-localize most with human FVIII. The splenic transcriptome highlights potential therapeutic targets to prevent inhibitors. SUMMARY: Background Developing factor VIII (FVIII) inhibitory antibodies is the most serious complication in hemophilia A treatment, representing a significant health and economic burden. A better understanding of the early events in an immune response leading to this outcome may provide insight into inhibitor development. Objective To identify early mediators of FVIII immunity and to detail immune expression profiles in the spleen and liver. Methods C57Bl/6 F8 E16 knockout mice were infused with 5-20 µg (2000-8000 IU kg-1 ) of recombinant FVIII. Spleens were frozen at various time-points post-infusion and stained for FVIII and cellular markers. Splenic and liver RNA expression analysis was performed 3 h post-infusion of 0.6 µg (240 IU kg-1 ) FVIII by nCounter technology using a 561-gene immunology panel. Results FVIII localization in the spleen did not change over 2.5 h. We observed significantly higher co-localization of FVIII with MARCO+ cells compared with SIGLEC1+ and SIGNR1+ in the splenic marginal zone. FVIII exhibited little co-localization with CD11c+ dendritic cells and the macrophage mannose receptor, CD206. Following FVIII infusion, the splenic mRNA profiling identified genes such as Tnfaip6 and Il23r, which are implicated in chemotaxis and a proinflammatory Th17 response, respectively. In contrast, an upregulation of Gfi1 in the liver suggests an anti-inflammatory environment. Conclusions FVIII co-localizes predominantly with marginal zone macrophages (MARCO+ ) in the murine spleen following intravenous infusion. Targeting pathways that are implicated in the early FVIII innate immune response in the spleen may lead to therapeutic interventions to prevent inhibitor formation.

Antigen presentation to CD8+ T cells: cross-priming in infectious diseases.

Recent studies indicate that, in most types of infections, antigen presentation by 'professional' bone-marrow-derived cells is essential for priming pathogen-specific CD8+ T cells. This is true even in the absence of direct infection of these cells, which indicates that cross-priming is an essential component of the immune response against pathogens.

Disturbed phase relations in white matter hyperintensity based vascular dementia: an EEG directed connectivity study.

OBJECTIVE: White matter hyperintensities (WMH), a feature seen on magnetic resonance imaging (MRI) and regarded to reflect small vessel disease, can lead to vascular dementia (WMH-VaD). In WMH-VaD, cognitive deficits typically consist of executive function disturbances, and reduced information processing speed, regarded as a result of cerebral hypoperfusion. We aimed to investigate whether this patient group has typical functional differences from controls. METHODS: Resting-state encephalography studies of 17 VaD patients and 17 age- and gender matched non-demented controls were analysed in the delta, theta, alpha1 and 2, and beta frequency bands. Undirected functional connectivity between electrodes was established with the Phase Lag Index (PLI) and directed functional connectivity with the directed Phase Lag Index (dPLI). PLI and dPLI were related to performance in cognitive testing. RESULTS: Mean PLI did not differ between patients and controls. In the control group dPLI showed anterior to posterior phase gradients in all bands except the delta band. In the VaD patient group this pattern was significantly different without a clear directional pattern. No relationship with cognition was demonstrated. CONCLUSIONS: This study shows a clear front-to-back direction of connectivity in non-demented controls. In VaD patients with extensive WMH, this pattern is disturbed. SIGNIFICANCE: Structural damage at the regions of long distance white matter tracts may induce changes in the direction of phase relationships of distinct brain regions.

Cholinergic receptor activation on epithelia protects against cytokine-induced barrier dysfunction.

AIM: Various types of cholinergic receptors are expressed on intestinal epithelia. Their function is not completely understood. We hypothesize that cholinergic receptor activation on epithelium may serve a protective function in cytokine-induced barrier dysfunction. METHODS: The effect of cholinergic receptor activation on cellular barrier function in epithelial cells was assessed by measuring electrical impedance, and by determining para-cellular transport in transwell experiments. Cell lysates treated with cytokine and/or cholinergic agonists were analysed for cyto- and chemokine production, and tight junction (TJ) protein rearrangement was assessed. Primary colonic epithelial cells were isolated from surgically resected colon tissue of patients with inflammatory bowel disease. RESULTS: IL-1ß induced production of chemokines (CXCL-1, CXCL-10, IL-8, CCL-7) and led to a rearrangement of TJ proteins (occludin and ZO-1). This response was inhibited by pre-treatment with muscarinic, rather than nicotinic, acetylcholine receptor agonists. Treatment with IL-1ß enhanced paracellular permeability (4kD dextran) and reduced impedance across the monolayer, which was counteracted by pre-incubation with acetylcholine, or muscarinic receptor agonist bethanechol. The protective effect of acetylcholine was antagonized by atropine, underscoring muscarinic receptor involvement. IL-1ß induced transcription of myosin light chain kinase and phosphorylation of myosin light chain, and this cytokine-induced phosphorylation of MLC was inhibited by muscarinic receptor agonists. Furthermore, in epithelial cells from resection material of patients with Crohn's disease and ulcerative colitis, high expression of CXCL-8 was associated with a reduced choline acetyl transferase expression, suggesting an aberrant epithelial production of ACh in inflammatory context. CONCLUSION: Acetylcholine acts on muscarinic receptors on epithelial cells to maintain epithelial barrier function under inflammatory conditions.

In vivo detection of fluorescent tumor-specific cytotoxic T cell clones.

Cytotoxic T lymphocytes (CTL) can be very effective mediators of tumor-specific immunity in vivo. Since little is known about the in vivo behaviour of cultured tumor-specific CTL, a fast and simple method has been developed utilizing a lipophilic carbocyanine, 1,1'-dioctadecyl 3,3,3',3'-tetramethylin-docarbocyanine perchlorate (DiI), for the in vivo detection of tumor-specific CTL clones in (tumor-bearing) mice. The two CTL clones used in this study are directed against human papillomavirus type 16- or human adenovirus type 5 early region 1 (Ad5E1)-transformed mouse embryo cells. Growth ability, cytotoxic capacity and tumor-eradicating potential remained unaltered when the CTL were labeled with this dye. Thus, in neither in vitro nor in vivo testing was the biological function of the CTL clones affected. The in vivo localization in the spleen of an adoptively transferred DiI-labeled Ad5E1-specific CTL clone is described. This adoptively transferred CTL clone was also detectable at the site of a subcutaneously growing human Ad5E1-induced tumor within 1 day after intravenous injection.

Preliminary studies on the interaction of TNF alpha and IFN gamma with alpha 2-macroglobulin.

The binding of 125I-labelled recombinant human TNF alpha and IFN gamma to isolated human blood alpha 2-macroglobulin has been investigated using molecular sieving procedures and non-denaturing PA gel electrophoresis in combination with autoradiography. These studies revealed that both cytokines readily bind to the electrophoretically fast form of alpha 2M generated by methylamine or protease treatment of this protein. PAGE/SDS gel investigations indicated that TNF alpha bound non-covalently while the IFN gamma interaction was covalent in nature. Preliminary competition studies also indicate that cold TNF alpha and IL-2 are more effective than cold IFN gamma at inhibiting the binding of labelled IFN gamma to alpha 2M. Bioassays revealed that "native" alpha 2M or its derivatives at 2 mg/ml concentration did not impair the antiproliferative effects of TNF alpha and IFN gamma on susceptible bladder tumour cell lines. Furthermore they did not interfere in the induction of Class II antigen expression by IFN gamma on inducible cell lines or in a 2-site ELISA assay for TNF.

Excreted/secreted Trichuris suis products reduce barrier function and suppress inflammatory cytokine production of intestinal epithelial cells.

The administration of helminths is considered a promising strategy for the treatment of autoimmune diseases due to their immunomodulatory properties. Currently, the application of the helminth Trichuris suis as a treatment for Crohn's disease is being studied in large multi-center clinical trials. The intestinal epithelium forms an efficient barrier between the intestinal lumen containing the microbial flora and helminths, and dendritic cells (DCs) present in the lamina propria that determine the TH response. Here, we investigated how excreted/secreted (E/S) products of T. suis affect the barrier function of intestinal epithelial cells (IECs) in order to reach the DCs and modulate the immune response. We show that T. suis E/S products reduce the barrier function and the expression of the tight junction proteins EMP-1 and claudin-4 in IEC CMT93/69 monolayers in a glycan-dependent manner. This resulted in an increased passage of soluble compounds to the basolateral side that affected DC function. In addition, T. suis E/S suppressed LPS-induced pro-inflammatory cytokine production by CMT93/69 cells, whereas the production of the TH2 response-inducing cytokine thymic stromal lymphopoietin (TSLP) was induced. Our studies indicate that T. suis E/S glycans affect the function of the intestinal epithelium in order to modulate DC function. Identification of the T. suis E/S glycans that modulate IEC and DC function may lead to a strategy to reduce symptoms of autoimmune and allergic immune diseases by orally administrated helminth-derived factors without the need of infection with live helminths.

Genomic identification of the minor histocompatibility antigen HA-1 locus by allele-specific PCR.

Graft-versus-host disease (GvHD) can be a major complication of allogeneic bone marrow transplantation even in recipients of HLA genotype-identical transplants. Disparities in minor histocompatibility antigens (mHags) between donor and recipient are a potential risk for the development of GvHD. A mismatch for the mHag HA-1 can cause GvHD in adult recipients of allogeneic bone marrow from HLA-identical donors. The mHag HA-1, first identified by HLA-A*0201-restricted cytotoxic T cells (CTLs), was recently chemically characterized as a nonapeptide. On the cDNA level, the HA-1 locus has two alleles, HA-1H and HA-1R, which differ in two nucleotides, resulting in a single amino acid substitution. Here we report on the genomic structure of the HA-1 locus. Isolation and sequencing of cosmid DNA encoding the HA-1 peptide sequence revealed that the HA-1 alleles are encoded by two exons. Two different primer sets were designed, each consisting of allele-specific primers and a common primer, and both sets containing intronic sequences. We performed genomic DNA typing of three families consisting of 24 HLA-A*0201-positive individuals. The predicted allele-specific products correlated in all cases with the mHag classification by CTLs and by RT-PCR. We demonstrate for the first time the genomic identification of the mHag HA-1 locus. Prospective genomic typing for the HA-1 alleles will improve donor selection and identify HLA-A*0201-positive recipients with a high risk for HA-1-induced GvHD.

Conservation of minor histocompatibility antigens between human and non-human primates.

It is well accepted that minor histocompatibility antigens (mHag) can function as transplantation barriers between HLA-matched individuals. Little is known about the molecular nature and evolutionary conservation of mHag. It is only very recently that the first human mHag were identified. The HLA-A2.1-restricted mHag HA-2 and the HLA-B7-restricted mHag H-Y appeared to be peptides derived from polymorphic self proteins. Here we show that the HLA-A2.1-restricted mHag HA-1, HA-2, and the H-Y peptides are conserved between man, chimpanzees and rhesus macaques. Human cytotoxic T cell clones specific for the HLA-A2.1-restricted mHag HA-1, HA-2, and H-Y recognized HLA-A2.1 gene-transfected chimpanzee and rhesus macaque cells. High-pressure liquid chromatography fractionation of HLA-A2.1-bound peptides isolated from the HLA-A2.1-transfected chimpanzee cells revealed that the chimpanzee HA-1 and HA-2 co-eluted with the human HA-1 and HA-2. Subsequent amino acid sequencing showed that the chimpanzee HA-2 peptide is identical to the human HA-2 peptide. Our functional and biochemical results demonstrate that mHag peptides are conserved for over 35 million years.

The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition.

A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.

Cutting edge: the HLA-A*0101-restricted HY minor histocompatibility antigen originates from DFFRY and contains a cysteinylated cysteine residue as identified by a novel mass spectrometric technique.

In this report, we describe the use of novel mass spectrometry instrumentation to identify a male-specific minor histocompatibility Ag restricted by HLA-A*0101 (A1-HY). This Ag has the sequence IVDC*LTEMY, where C* represents a cysteine disulfide bonded to a second cysteine residue. The core peptide sequence is found in the protein product of DFFRY, a Y chromosome gene not previously identified as the source of an HY Ag. The male-specific form of the peptide differs from its X chromosomal counterpart by the substitution of serine for the C* residue. Both peptides are expressed on the cell surface at 30 or fewer copies per cell. However, A1-HY-specific CTL recognize the DFFRY-derived peptide at a 1500-fold lower dose than the female homologue. Thus, these studies have identified a new source of HY epitopes and provide additional information about the influence of posttranslational modifications of class I-associated peptides on T cell recognition.