Studying bicyclists' perceived level of safety using a bicycle simulator combined with immersive virtual reality.

There is a need for methods that provide a better understanding of bicyclists' perceived safety and preferences on currently unavailable and/or unknown bicycle facilities. Different survey methods have been used to study bicyclists' behavior, experiences, and preferences; ranging from verbally described facilities to surveys including images and videos. Virtual Reality (VR) experiments blur the boundaries between stated preference (SP) surveys and revealed preference (RP) surveys and provide a realistic sense of design. This research introduces a novel research method in bicycling research and discusses the results of an experiment using a bicycle simulator combined with immersive VR. In total, 150 participants participated in this experiment and were asked about demographics and perceptions and preferences after bicycling in five different environments with an instrumented bicycle in VR. A 5 × 2 mixed design was used with bicycling environment as within-subject factor and pedestrian / traffic volume as between-subject factor. ANOVA tests revealed how each environment and ambient pedestrian / traffic volume affected perceived level of safety (PLOS) and willingness to bicycle (WTB). Pairwise comparison showed that participants felt safer bicycling on the segregated bicycle path compared to bicycling on the painted bicycle path on the road and roadside. There was no meaningful difference between WTB for less than 10 min and WTB for more than 10 min between bicycling on a painted bicycle path on the sidewalk and painted bicycle path on the road. PLOS and WTB ratings of men and women were not significantly different from each other. The older segment of the sample was more worried about roadside bicycling and bicycle commuters were more confident to ride on the roadside. Despite having several limitations, immersive 360-degree VR was found a powerful presentation tool to evaluate future street designs which can inform transport and urban planning.

Type III bare lymphocyte syndrome associated with a novel RFXAP mutation: a case report.

Type III bare lymphocyte syndrome (BLS) is a severe combined immunodeficiency disease caused by the absence of MHC Class II expression associated with low expression of class I molecules. Here, we report a case with type III BLS who lacked RFXAP (Regulatory factor X-associated protein) expression as a result from a novel mutation introducing a premature stopcodon in DE-region at amino acid 73.

T cell immune reconstitution after allogeneic bone marrow transplantation in bare lymphocyte syndrome.

To study the impact of an MHC class II-negative environment on T cell immune reconstitution, we have analyzed the phenotypical and functional characteristics of FACS-sorted cultured CD4(+) and CD8(+) T cells in two Bare Lymphocyte Syndrome (BLS) patients before and after allo-BMT. A similar analysis was performed in two MHC class II expressing pediatric leukemia patients after treatment with an allo-BMT who were included in our study as control. It was observed that CD4(+) T cells displayed cytolytic alloreactivity in both BLS patients prior to and within the first year after allo-BMT, whereas such cells were absent at a later time-point, in the donors and pediatric leukemia controls. In addition, reduced MHC class II expression was observed in CD8(+) T cells of both recipients early after allo-BMT, irrespective of the T cell chimerism pattern. Lack of endogenous MHC class II expression in BLS patients, therefore, results in aberrant T cell selection within the first year after allo-BMT, analogous to T cell selection before transplantation. These T cell selection processes seem to be normalized at a later time point after allo-BMT probably due to migration and integration of graft-derived MHC class II-positive antigen presenting cells to sites of T cell selection.

Defective MHC class II expression in an MHC class II deficiency patient is caused by a novel deletion of a splice donor site in the MHC class II transactivator gene.

MHC class II deficiency patients are mutated for transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. Four complementation groups (A-D) are defined and the gene defective in group A has been shown to encode the MHC class II transactivator (CIITA). Here, we report the molecular characterization of a new MHC class II deficiency patient, ATU. Cell fusion experiments indicated that ATU belongs to complementation group A. Subsequent mutation analysis revealed that the CIITA mRNA lacked 84 nucleotides. This deletion was the result of the absence of a splice donor site in the CIITA gene of ATU. As a result of this novel homozygous genomic deletion, ATU CIITA failed to transactivate MHC class II genes. Furthermore, this truncated CIITA of ATU did not display a dominant negative effect on CIITA-mediated transactivation of various isotypic MHC class II promoters.

Transcriptional control of MHC genes and T cell development in MHC class II deficiency.

MHC class II deficiency has proven to be an excellent model to study transcription regulation of MHC genes and T cell development. Cell lines established from MHC class II deficient patients have been of great value for the identification of proteins necessary for MHC expression and their study has resulted in the identification of a common regulatory pathway for MHC class II and class I genes. The lack of MHC class II expression was found to have a profound effect on the development of the CD4+ T cell lineage, in particular on the composition of the T cell receptor repertoire, revealing aberrant thymic selection processes in these patients. Here, we will discuss several aspects of the transcriptional regulation of MHC genes and the impact of deficient MHC class II expression on T cell development.

Discoordinate expression of invariant chain and MHC class II genes in class II transactivator-transfected fibroblasts defective for RFX5.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific transcription factors. In the present study, we show that fibroblasts derived from a recently identified bare lymphocyte syndrome patient, SSI, were mutated for RFX5, one of the DNA-binding components of the RFX complex. Despite the lack of functional RFX5 and resulting MHC class II-deficient phenotype, transfection of exogenous class II transactivator (CIITA) in these fibroblasts can overcome this defect, resulting in the expression of HLA-DR, but not of DP, DQ, and invariant chain. The lack of invariant chain expression correlated with lack of CIITA-mediated transactivation of the invariant chain promoter in transient transfection assays in SSI fibroblast cells. Consequently, these CIITA transfectants lacked Ag-presenting functions.

Incomplete T-cell immune reconstitution in two major histocompatibility complex class II-deficiency/bare lymphocyte syndrome patients after HLA-identical sibling bone marrow transplantation.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II-deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8(+) T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II-expressing CD4(+) and in the CD8(+) T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.

Molecular analysis of an MHC class II deficiency patient reveals a novel mutation in the RFX5 gene.

Patients suffering from major histocompatibility complex (MHC) class II deficiency, a rare primary immunodeficiency, are characterized by a lack of MHC class II expression which is the result of defects in trans-acting factors. At least four complementation groups, A, B, C, and D, can be discerned. The gene affected in group C patients is known to be RFX5 and encodes one of the subunits of the multimeric phosphoprotein complex, RFX. In the present study we fused fibroblasts of a recently identified MHC class II deficiency patient, OSE, with fibroblasts derived from patients representative of each of the four complementation groups. Transient heterokaryon analysis indicated that OSE belonged to complementation group C. Furthermore, transfection of wild-type RFX5 cDNA into OSE fibroblasts resulted in restoration of the defect. Mutation analysis revealed that the RFX5 mRNA lacked four nucleotides and that this deletion was the consequence of a G to A transition in a splice acceptor site. Genomic oligotyping demonstrated that OSE was homozygous for the splice site mutation.

The RFX complex is crucial for the constitutive and CIITA-mediated transactivation of MHC class I and beta2-microglobulin genes.

In type III bare lymphocyte syndrome (BLS) patients, defects in the RFX protein complex result in a lack of MHC class II and reduced MHC class I cell surface expression. Using type III BLS cell lines, we demonstrate that the RFX subunits RFX5 and RFXAP are crucial for constitutive and CIITA-induced MHC class I and beta2m transactivation. Similar to MHC class II, the promoters of MHC class I and beta2m contain an S-X-Y region of which the X1 box is crucial for constitutive and CIITA-induced MHC class I and beta2m transactivation. Thus, the RFX complex is part of a regulatory pathway linking the transactivation of MHC class I and II and their accessory genes.

Regulation of MHC class I and II gene transcription: differences and similarities.

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes.

Introduction of exogenous class II trans-activator in MHC class II-deficient ABI fibroblasts results in incomplete rescue of MHC class II antigen expression.

Previously, we have shown that fibroblasts established from type III bare lymphocyte syndrome patient ABI are characterized by the absence of MHC class II gene expression and a strongly reduced amount of MHC class I transcripts. Complementation analysis has suggested that the gene defective in these ABI fibroblasts is different from that encoding the class II trans-activator (CIITA), which has been attributed an essential role in both constitutive and inducible expression of MHC class II genes. In the present study it is shown by reverse transcriptase-PCR analysis that the amount of CIITA transcripts in ABI fibroblasts is greatly reduced compared with that in fibroblasts derived from a healthy individual. Transient cotransfection of a construct in which CIITA is under the control of a constitutive promoter with an HLA-DRA promoter-luciferase reporter plasmid resulted in enhanced luciferase expression in ABI fibroblasts. Furthermore, ABI fibroblasts stably transfected with CIITA re-express functional HLA-DR Ags, but do not express HLA-DQ and DP Ags at the cell surface. Comparison of these data with those obtained for normal fibroblasts and fibroblasts defective for CIITA indicate that the gene defect and the resulting lack of MHC class II expression in ABI fibroblasts can only partly be corrected by the introduction of CIITA. Furthermore, DNase I hypersensitivity analysis of ABI fibroblasts has revealed a closed chromatin structure in the promoter region of the MHC class II DRA gene. However, CIITA transfection resulted in an open DNA configuration, which suggests a role for CIITA in provoking changes in the chromatin structure of the DRA gene.

Selective usage of defined TCRBV genes in response to filarial antigens.

The characterization of T cell reactivities that are prone to down-modulation by filarial parasites is central to understanding how these nematodes can survive for long periods of time within their human host and to design appropriate immunoprophylactic measures. In the present study, TCRBV gene usage was analyzed in response to filarial antigens by PCR using a panel of TCRBV gene segment family-specific oligonucleotide primers. Analysis of individuals highly responsive to Brugia malayi adult worm antigen (BmA) (n = 4) indicated that following stimulation with BmA a maximum of four TCRBV gene families were over-represented in each subject. Those were TCRBV2, 9, 19 and 23 in subject 1; TCRBV8, 9 and 16 in subject 2; TCRBV2, 8, 9 and 11 in subject 3; and TCRBV13 and 23 in subject 4. The analysis of one subject who was unresponsive to BmA before but regained responsiveness after diethylcarbamazine treatment revealed that there was no overexpression of a particular TCRBV gene family before chemotherapy, whereas after chemotherapy three TCRBV gene families (TCRBV8, 16 and 19) were found to be overexpressed. Complementarity determining region 3 size analysis of a selection of the overexpressed TCRBV genes displayed oligoclonality in some of the observed expansions. Together these observations show that limited T cell subpopulations are clonally amplified in BmA-stimulated peripheral blood mononuclear cells of filarial responder subjects, possibly driven by a restricted number of antigens.

Human T cell repertoire generation in the absence of MHC class II expression results in a circulating CD4+CD8- population with altered physicochemical properties of complementarity-determining region 3.

In this study, we have investigated the impact of deficient MHC class II expression on the use of TCRBV6 and TCRBJ gene elements, and on the pattern of amino acid incorporation exhibited in the N1-D-N2 segments of the third complementarity-determining region (CDR3) of these TCRBV6 rearrangements. To this end, we have analyzed circulating T cells from three, nonrelated MHC class II-deficient (bare lymphocyte syndrome (BLS)) patients and three MHC class II-expressing family members. The patients and healthy controls exhibited similar, nonrandom usage profiles of TCRBV6 and TCRBJ gene elements in both the CD4+CD8- and the CD4-CD8+ subsets of peripheral blood T cells. No statistically significant differences between patients and controls were detected in the length of CDR3, or in the amount of non-germline modification at the sites of recombination. However, detailed analysis of the TCRBV6 rearrangements derived from the CD4+CD8- subsets from the BLS patients revealed patterns of amino acid incorporation into the N1-D-N2 region of CDR3 that resulted in altered charge and hydropathicity properties of the presumed Ag binding site. In this way, we have been able to demonstrate that human T cell repertoire development in the absence of MHC class II expression results in a circulating CD4+CD8- T cell population bearing TCRs with altered CDR3 profiles. Such altered profiles are likely to be a direct reflection of the lack of MHC class II-mediated selection processes in these BLS patients.

T cell development in a major histocompatibility complex class II-deficient patient.

In this report we show that the major histocompatibility complex (MHC) class II-negative thymus of a bare lymphocyte syndrome (BLS) patient contains a reduced CD4+ CD8- T cell population when compared to thymocytes derived from a MHC class II-expressing thymus. Of these CD4+ CD8- BLS thymocytes, approximately only one third co-expressed the CD3 antigen, moreover at a lower expression level when compared to control thymocytes. This suggests a partial maturation of the CD4+ CD8- T cells in the absence of MHC class II expression. Among the BLS thymocytes, CD4+ CD8+ thymocytes could easily be detected. Noteworthy, the number of CD4- CD8+ thymocytes was significantly increased. CD4+ CD8- T cells could also be found among the BLS peripheral blood mononuclear cells, albeit at reduced numbers. Despite the absence of peripheral MHC class II expression, the majority of these CD4+ CD8- T cells co-expressed the CD45RO marker. In the BLS patient, thymocytes as well as peripheral CD4+ CD8- T cells were not restricted in the use of the available T cell receptor (TcR) V gene family pool. However, the lack of detectable levels of thymic and peripheral MHC class II antigen expression in the BLS patient had altered the CD4-skewing patterns of TcR V gene families which were present in normal individuals. In conclusion, the lack of MHC class II expression in the BLS patient does not completely inhibit the CD4+ CD8- T cell development.

Lymphokine gene transcription in CD4+CD8- T cells of a type III bare lymphocyte syndrome patient.

In this study we analyzed the impact of a MHC class-II-deficient environment on the differentiation of CD4+CD8- T lymphocytes into functional defined subsets of lymphokine-producing T-helper cells. To this end a CD4+CD8- T-cell line and CD4+CD8- T-cell clones, isolated from PBMCs of a type III BLS patient, were stimulated in vitro with anti-CD3 and PMA and assessed for lymphokine transcription patterns. The results of these analyses show that CD4+CD8- T cells that have matured in a MHC class-II-deficient environment display lymphokine transcription patterns that resemble those of MHC class-II-expressing family control-derived CD4+CD8- T cells.

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8- peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8- T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V alpha and V beta amplimers, specific for each of the most known V alpha- and V beta-gene region families. The results of our studies demonstrate that some of the V alpha-gene segments are used less frequent in the CD4+CD8- T-cell subset of the patient, whereas the majority of the TCR V alpha- and V beta-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V alpha 12 transcripts was greatly diminished in the patient, both in the CD4+CD8- as well as in the CD4-CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8- T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V alpha-gene segments toward the CD4-CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Analysis of the peripheral T-cell compartment in the MHC class II deficiency syndrome.

To analyse the impact of lack of MHC class II expression on the composition of the peripheral T-cell compartment in man, the expression characteristics of several membrane antigens were examined on peripheral blood lymphocytes (PBL) and cultured T cells derived from an MHC-class-II-deficient patient. No MHC class II expression could be detected on either PBL or activated T cells. Moreover, the expression of MHC class I was reduced both on PBL and in vitro activated T cells compared to the healthy control. However, the reduced expression of CD26 observed on the PBL of the patient was restored after in vitro expansion. Despite the presumably class-II-deficient thymic environment, a distinct but reduced single CD4+ T-cell population was observed in the PBL of the patient. After in vitro expansion, the percentage of CD4+ cells dropped even further, most likely due to a proliferative disadvantage, compared to the single CD8+ T-cell population. However, proliferation analysis showed that T-cell activation via the TcR/CD3 pathway is not affected by the MHC class II deficiency.