IL-4 and IL-13 alter plasmacytoid dendritic cell responsiveness to CpG DNA and herpes simplex virus-1

Human plasmacytoid dendritic cells (pDCs) are found in skin lesions in a wide variety of diseases. The role of the microenvironment in these lesions on the function of human pDCs remains elusive. We sought to determine the effect of T(h)2 cytokines on the ability of human pDCs to respond to CpG oligodeoxynucleotides (ODNs) and herpes simplex virus in vitro. In this study, we found that the T(h)2 cytokines, IL-4 and IL-13, modulate Toll-like receptor 9 (TLR-9)- and herpes simplex virus-induced pDC phenotype and enhance the ability of these cells to induce allogeneic T-cell responses. Moreover, T(h)2 cytokines impaired TLR-9-induced secretion of inflammatory cytokines and chemokines. Taken together, these results demonstrate that T(h)2 cytokines are involved in the modulation of pDC function and responsiveness to bacterial- and viral-derived stimuli.     

Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell�CAssociated Epitopes Using Combinatorial MHC Multimers

OBJECTIVE

Type 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8⁺ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8⁺ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive.

RESEARCH DESIGN AND METHODS

Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B_10–18, prepro-insulin (PPI)_15–24, islet antigen (IA)-2_797–805, GAD65_114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)_265–273, and prepro islet amyloid polypeptide (ppIAPP)_5–13–specific CD8⁺ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients.

RESULTS

Using this kit, islet autoreactive CD8⁺ T-cells recognizing insulin B_10–18, IA-2_797–805, and IGRP_265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI_15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome.

CONCLUSIONS

A kit was developed that allows simultaneous detection of CD8⁺ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.Type 1 diabetes results from a selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. It is becoming increasingly clear that islet epitope–specific CD8⁺ T-cells play a pivotal role in the destruction process and constitute a significant portion of insulitis (1,2). In accordance, nonobese diabetic mice lacking the expression of major histocompatibility complex (MHC) class I are resistant to autoimmune diabetes (3,4), whereas HLA-A2 transgenic nonobese diabetic mice develop accelerated disease (5). Additionally, transfer of CD8⁺ T-cell clones resulted in transfer of type 1 diabetes (6,7). Thus, detection and monitoring of specific CD8⁺ T-cells may provide a valuable tool to assess the disease activity.Islet cell transplantation has considerable potential as a cure for type 1 diabetes (8). Several groups have reported short-term success, using different islet isolation and immunosuppressive regimens (9–12), but long-term insulin independence is rare (13). The rationale behind transplantation of islet cells is replenishment of destructed cells. Yet, as the insulin-producing cells were destructed by an autoimmune response, islet cell transplantation could also result in reactivation of the autoimmune response. Recently, we have shown that proliferation of CD4⁺ T-cells specific for GAD and IA-2 in patients who underwent islet cell transplantation is associated with clinical outcome (14). Yet, ultimately, the destruction of β-cells is likely to be caused by CD8 T-cells.The epitopes recognized by the diabetes-specific human autoreactive CD8⁺ T-cells are primarily derived from β-cell antigens, most importantly (pre-)(pro-)insulin. Previously, we showed that the presence of CD8⁺ T-cells reactive to the naturally processed insulin–peptide B_10–18 in HLA-A2 correlated with islet cell destruction (15). Recently, another important epitope that was uncovered as the signal peptide of pro-insulin was shown to contain a glucose-regulated CD8⁺ T-cell epitope (prepro-insulin [PPI]_15–24) (16), but many other epitopes derived from insulin and a range of other β-cell–derived antigens, such as GAD65 (17), islet antigen (IA)-2 (18), islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) (19,20), and prepro islet amyloid polypeptide (ppIAPP) (21), have been reported (rev. in 22). Ideally, monitoring for the presence of CD8⁺ T-cells reactive to all of the above-mentioned epitopes simultaneously would be desired, posing considerable constraints on blood volumes accessible for monitoring of islet autoimmunity with conventional immune assays.Currently, monitoring of CD8⁺ T-cells reactive to β-cell–derived antigens requires staining of a large number of, usually fresh, cells with HLA tetramers loaded with a single peptide, or in vitro culture for functional immune assays (proliferation, cytokine production [ELISPOT]). Monitoring multiple epitope-specific CD8⁺ T-cell populations by conventional tetramer technology is generally impossible because of the scarcity of material. Furthermore, detection of islet autoreactive T-cells is hampered by their low precursor frequencies in circulation (23,24), low T-cell receptor (TCR) avidity (15), potentially low binding affinity of peptide epitopes to HLA (25), a wide range of candidate islet epitopes (22), and the existence of regulatory T-cells (26,27).Therefore, we used the recently described combinatorial quantum dot (Qdot) technique (28) to simultaneously detect CD8⁺ T-cells specific for six different β-cell–derived antigens, a naturally occurring HLA-A2 derived peptide, and a mix of viral epitopes in HLA-A2 multimers. Using peripheral blood cells from recent-onset type 1 diabetic patients, their siblings, and control subjects, we validated this technique and established the specificity of these stainings. Subsequently, we monitored the presence of reactive CD8⁺ T-cells before and at several time points after clinical islet cell transplantation. Altogether, we developed a high-throughput and relatively sensitive and specific Diab-Q-kit, allowing simultaneous detection of autoreactive CD8⁺ T-cells to multiple islet epitopes, which is applicable to small volumes of stored blood samples to allow screening in multicenter immune intervention trials.     

Explaining service use for mental health problems in the Dutch general population: the role of resources, emotional disorder and functional impairment

Objective To analyse explanations of service use in terms of resources, emotional (mood or anxiety) disorder and functional impairment. Method Data was derived from a prospective cohort study in a sample representative (n = 4848) of the Dutch adult general population. Results The occurrence of an emotional (mood or anxiety) disorder led to a greater use of services as a partial consequence of the functional impairments that accompanied the disorder, but this applied only to primary care services and not to specialised mental health services. After adjustment for the influence of all other determinants in the model, people with more education and those with higher neuroticism scores were more likely to use specialised services in particular. Conclusions Future research could benefit from applying the models derived here to further clarify the use of the two service modalities, as well as to assess additional psychological resources.     

External quality assessment program for qualitative and quantitative detection of hepatitis C virus RNA in diagnostic virology

To assess the performance of laboratories in detecting and quantifying hepatitis C virus (HCV) RNA levels in HCV-infected patients, we distributed two proficiency panels for qualitative and quantitative HCV RNA testing. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each panel consisted of two negative samples and six positive samples, with HCV RNA target levels from 200 to 500,000 copies/ml. Panel 1 had four samples with at least 50,000 copies/ml, and panel 2 had two samples with at least 50,000 copies/ml. Fifty-seven laboratories submitted 45 qualitative and 35 quantitative data sets on panel 1, and 81 laboratories submitted 75 qualitative and 48 quantitative data sets on panel 2. In both panels, about two-thirds of the qualitative data sets and >90% of the quantitative data sets were obtained with commercial assays. With each panel, two data sets gave one false-positive result, corresponding to false-positivity rates of 1.3% and 0.8% for panel 1 and panel 2, respectively. Samples containing at least 50,000 copies/ml were found positive in 97% and 99% of the cases with panel 1 and panel 2, respectively. In contrast, the positive samples containing < or =5,000 copies/ml were reported positive in only 71% and 77% of the cases with panel 1 and panel 2, respectively. Adequate or better scores on qualitative results (all results correct or only the low-positive samples missed) were obtained in 84% (panel 1) and 80% (panel 2) of the data sets. In the analysis of quantitative results, 60% (panel 1) and 73% (panel 2) of the data sets obtained an adequate or better score (> or =80% of the positive results within the range of the geometric mean +/- 0.5 log(10)). Our results indicate that considerable improvements in molecular detection and quantitation of HCV have been achieved, particularly through the use of commercial assays. However, the lowest detection levels of many assays are still too high, and further standardization is still needed. Finally, this study underlines the importance of proficiency panels for monitoring the quality of diagnostic laboratories.     

Microradiography to evaluate bone growth into a rat mandibular defect

Microradiography has been evaluated to measure bone healing into a 5.0mm outer diameter mandibular defect in the rat. This method provides high-resolution radiographs of the defects that can be used for an accurate measurement of bone defect healing. In 12 rats, the defect widths of 42-day-old mandibular defects have been measured both using microradiographs and histological sections. The defect width+/-S.D. measured 3.42+/-0.98 mm microradiographically and 3.47+/-1.11 mm histologically. Both methods were accurate in determining defect widths but microradiography has the advantage over histology that an image is obtained from the entire defect, making it possible to measure areas of bone growth.