Generation and Study of Antibodies against Two Triangular Trimers Derived from Aß.

Monoclonal antibodies (mAbs) that target the P-amyloid peptide (Aß) are important Alzheimer's disease research tools and are now being used as Alzheimer's disease therapies. Conformation-specific mAbs that target oligomeric and fibrillar Aß assemblies are of particular interest, as these assemblies are associated with Alzheimer's disease pathogenesis and progression. This paper reports the generation of rabbit mAbs against two different triangular trimers derived from Aß. These antibodies are the first mAbs generated against Aß oligomer mimics in which the high-resolution structures of the oligomers are known. We describe the isolation of the mAbs using single B-cell sorting of peripheral blood mononuclear cells (PBMCs) from immunized rabbits, the selectivity of the mAbs for the triangular trimers, the immunoreactivity of the mAbs with aggregated Aß42, and the immunoreactivity of the mAbs in brain tissue from the 5xFAD Alzheimer's disease mouse model. The characterization of these mAbs against structurally defined trimers derived from Aß enhances understanding of antibody-amyloid recognition and may benefit the development of diagnostics and immunotherapies in Alzheimer's disease.

Antibodies Raised Against an Aß Oligomer Mimic Recognize Pathological Features in Alzheimer's Disease and Associated Amyloid-Disease Brain Tissue.

Antibodies that target the ß-amyloid peptide (Aß) and its associated assemblies are important tools in Alzheimer's disease research and have emerged as promising Alzheimer's disease therapies. This paper reports the creation and characterization of a triangular Aß trimer mimic composed of Aß17-36 ß-hairpins and the generation and study of polyclonal antibodies raised against the Aß trimer mimic. The Aß trimer mimic is covalently stabilized by three disulfide bonds at the corners of the triangular trimer to create a homogeneous oligomer. Structural, biophysical, and cell-based studies demonstrate that the Aß trimer mimic shares characteristics with oligomers of full-length Aß. X-ray crystallography elucidates the structure of the trimer and reveals that four copies of the trimer assemble to form a dodecamer. SDS-PAGE, size exclusion chromatography, and dynamic light scattering reveal that the trimer also forms higher-order assemblies in solution. Cell-based toxicity assays show that the trimer elicits LDH release, decreases ATP levels, and activates caspase-3/7 mediated apoptosis. Immunostaining studies on brain slices from people who lived with Alzheimer's disease and people who lived with Down syndrome reveal that the polyclonal antibodies raised against the Aß trimer mimic recognize pathological features including different types of Aß plaques and cerebral amyloid angiopathy.

Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

Upcycling the anthracyclines: New mechanisms of action, toxicology, and pharmacology.

The anthracyclines are a family of natural products isolated from soil bacteria with over 2000 chemical representatives. Since their discovery seventy years ago by Waksman and co-workers, anthracyclines have become one of the best-characterized anticancer chemotherapies in clinical use. The anthracyclines exhibit broad-spectrum antineoplastic activity for the treatment of a variety of solid and liquid tumors, however, their clinical use is limited by their dose-limiting cardiotoxicity. In this review article, we discuss the toxicity of the anthracyclines on several organ systems, including new insights into doxorubicin-induced cardiotoxicity. In addition, we discuss new medicinal chemistry developments in the biosynthesis of new anthracycline analogs and the synthesis of new anthracycline analogs with diminished cardiotoxicity. Lastly, we review new studies that describe the repurposing of the anthracyclines, or "upcycling" of the anthracyclines, as anti-infective agents, or drugs for niche indications. Altogether, the anthracyclines remain a mainstay in the clinic with a potential new "lease on life" due to deeper insight into the mechanism underlying their cardiotoxicity and new developments into potential new clinical indications for their use. Keywords: Anthracycline, chemotherapy, toxicology, medicinal chemistry, biosynthesis.

A BioBricks toolbox for metabolic engineering of the tetracenomycin pathway.

BACKGROUND/GOAL/AIM: The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. MAIN METHODS AND MAJOR RESULTS: We developed a BioBricks genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: (1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a "metabolic regulatory switch" responsible for mobilizing triacylglycerols to ß-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. CONCLUSIONS AND IMPLICATIONS: Altogether, this system is compatible with the BioBricks [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE. This production platform improves access to potent analogs, such as tetracenomycin X, and sets the stage for the production of new tetracenomycins via combinatorial biosynthesis.

How do we implement pathogen reduction technology, while maintaining an adequate platelet inventory for our patients?

Despite the significantly reduced infectious disease risk through robust and sensitive laboratory assays, comprehensive donor screening and good manufacturing practices, new and emerging infectious agents and bacterial contamination continue to pose a threat to the blood supply. Pathogen Reduction (PR) technology is an option to mitigate the risk of platelet transfusion transmitted infections. Here we describe our structure and strategies to implement PR technology. Pre-implementation and phased approach implementation processes from our hospital-based donor center, components processing laboratory, transfusion service, clinicians, nursing, and patient perspectives are described. Communication and reassessment of collection settings occurred between the donor center and components processing laboratory (CPL). During Phase 1, CPL consistently processed approximately 56% of monthly apheresis platelets (AP) collections by PR and the remaining 44% as conventional platelets (CP). Phase 2 increased the amount of AP undergoing PR from 56% to approximately 78%. A phased implementation and maintenance of a dual inventory may provide flexibility to blood collection, blood manufacturing, and transfusion service processes. Our dual inventory of PR and CP allows our transfusion service a readily available platelet inventory. A collaborative hospital-based donor center, component processing laboratory, and transfusion service are essential to the productivity and maintenance of the dual platelet inventory.

Determinants of Hip and Knee Replacement: The Role of Social Support and Family Dynamics.

The objective of this study was to examine variations in the determinants of joint replacement (JR) across gender and age, with emphasis on the role of social support and family dynamics. We analyzed data from the US Health and Retirement Study (1998-2010) on individuals aged 45 or older with no prior receipt of JR. We used logistic regression to analyze the probability of receiving knee or hip replacement by gender and age (<65, 65+). We estimated the effect of demographic, health needs, economic, and familial support variables on the rate of JR. We found that being married/partnered with a healthy spouse/partner is positively associated with JR utilization in both age groups (65+ group OR: 1.327 and <65 group OR: 1.476). While this finding holds for men, it is not statistically significant for women. Among women younger than 65, having children younger than 18 lowers the odds (OR: 0.201) and caring for grandchildren increases the odds (1.364) of having a JR. Finally, elderly women who report availability of household assistance from a child have higher odds of receiving a JR as compared with elderly women without a child who could assist (OR: 1.297). No effect of available support from children was observed for those below 65 years old and elderly men. Our results show that intrafamily dynamics and familial support are important determinants of JR; however, their effects vary by gender and age. Establishing appropriate support mechanisms could increase access to cost-effective JR among patients in need of surgery.

The Effects of Revision Total Hip Arthroplasty on Medicare Spending and Beneficiary Outcomes: Implications for the Comprehensive Care for Joint Replacement Model.

BACKGROUND: After the first year in the Comprehensive Care for Joint Replacement (CJR) model, hospitals must repay Medicare for spending above a target price. Hospitals are incentivized to reduce spending in a 90-day episode and generate internal cost savings through, for example, the use of lower-cost implants. METHODS: We used a Markov model to compare quality-adjusted life-years and lifetime costs of total hip arthroplasty, under Medicare fee-for-service (baseline) and under alternative revision rate assumptions (prospective CJR scenarios). Results were generated for 65-year-old and 75-year-old male and female Medicare beneficiaries using baseline spending and revision rates from Medicare claims. We estimated the impact of CJR on 90-day spending. We ran sensitivity analyses for revision rates. RESULTS: Under willingness-to-pay thresholds of $50,000, $100,000, and $150,000, the baseline scenario was more cost-effective than the CJR scenario for a 65-year-old male patient if the revision risk increases by at least 7% (95% confidence interval for CJR savings: 4%-22%), 5% (range, 3%-7%), or 3% (range, 1%-5%), respectively. For males aged 75 years and females, revision risk needs to increase by a greater percentage under CJR relative to baseline for Medicare fee-for-service to be more cost-effective. CONCLUSION: The CJR model holds great promise. However, it incentivizes hospitals to choose lower-cost implants and adopt newer technology more slowly, which could potentially increase revision rates and offset benefits of the program. Policy makers should monitor revision rates and consider changes to the CJR model to ensure beneficiary access to valuable technology.

Hospital Readmission Rates Following AMI: Potential Interventions to Improve Efficiency.

OBJECTIVES: Quality of care utilization measures for patients admitted to the hospital with an acute myocardial infarction (AMI) include length of stay (LOS) and 30-day readmission rates. Our aim was to test whether efforts resulting in reduced LOS in patients diagnosed as having AMI would result in a higher risk of readmission within 30 days of hospital discharge and whether specific interventions could be targeted to reduce readmissions. METHODS: Using data supplied by the Veterans Affairs Inpatient Evaluation Center, we analyzed both the readmissions within 30 days of an AMI and LOS and determined the timing of readmissions and associated diagnoses. RESULTS: During 2013-2015, 35 (13.3%) of 263 patients with AMI were readmitted within 30 days of discharge compared with 19 (13.4%) of 142 patients during 2016 (not significant). During the same time, LOS was <3 days in most patients. From 2013 to 2015, the initial hospital time was 6 ± 6 days, whereas time out of the hospital before readmission was 11 ± 8 days; these times did not differ from 2016. Initial therapeutic decisions were based on coronary anatomy in >90% of patients with a decision to proceed with revascularization in most patients. Diagnoses during readmission to the hospital were also similar during early and later time periods and most frequently were a result of either coronary artery bypass grafting-related complications from the initial hospitalization or elective coronary artery bypass grafting. Acute coronary syndrome-related diagnoses and recurrent noncardiac causes of chest pain also were common diagnoses during both time periods and did not involve extensive workup during the readmission. CONCLUSIONS: Readmissions for patients with AMI were stable during a 4-year period, at a time that efforts to reduce LOS were emphasized. Because a significant proportion of readmissions involved noncardiac sources of chest pain, improved communication between the emergency department and in-patient cardiology services at the time of triage may be a feasible way to improve efficiency of utilization.

Assessing the Utilization of Total Ankle Replacement in the United States.

BACKGROUND: Total ankle arthroplasty (TAR) has been shown to be a cost-effective procedure relative to conservative management and ankle arthrodesis. Although its use has grown considerably over the last 2 decades, it is less common than arthrodesis. The purpose of this investigation was to analyze the cost and utilization of TAR across hospitals. METHODS: Our analytical sample consisted of Medicare claims data from 2011 and 2012 for Inpatient Prospective Payment System hospitals. Outcome variables of interest were the likelihood of a hospital performing TAR, the volume of TAR cases, TAR hospital costs, and hospital profit margins. Data from the 2010 Cost Report and Medicare inpatient claims were utilized to compute average margins for TAR cases and overall hospital margins. TAR cost was calculated based on the all payer cost-to-charge ratio for each hospital in the Cost Report. Nationwide Inpatient Sample data were used to generate descriptive statistics on all TAR patients across payers. RESULTS: Medicare participants accounted for 47.5% of the overall population of TAR patients. Average implant cost was $13 034, accounting for approximately 70% of the total all-payer cost. Approximately, one-third of hospitals were profitable with respect to primary TAR. Profitable hospitals had lower total costs and higher payments leading to a difference in profit of approximately $11 000 from TAR surgeries between profitable and nonprofitable hospitals. No difference was noted with respect to length of stay or number of cases performed between profitable and nonprofitable hospitals. TAR surgeries were more likely to take place in large and major teaching hospitals. Among hospitals performing at least 1 TAR, the margin on TAR cases was positively associated with the total number of TARs performed by a hospital. CONCLUSION: There is an overall significant financial burden associated with performing TAR with many health systems failing to demonstrate profitability despite its increased utilization. While additional factors such as improved patient outcomes may be driving utilization of TAR, financial barriers may exist that can affect utilization of TAR across health systems. LEVEL OF EVIDENCE: Level III, comparative study.

Estimating the Societal Benefits of THA After Accounting for Work Status and Productivity: A Markov Model Approach.

BACKGROUND: Demand for total hip arthroplasty (THA) is high and expected to continue to grow during the next decade. Although much of this growth includes working-aged patients, cost-effectiveness studies on THA have not fully incorporated the productivity effects from surgery. QUESTIONS/PURPOSES: We asked: (1) What is the expected effect of THA on patients' employment and earnings? (2) How does accounting for these effects influence the cost-effectiveness of THA relative to nonsurgical treatment? METHODS: Taking a societal perspective, we used a Markov model to assess the overall cost-effectiveness of THA compared with nonsurgical treatment. We estimated direct medical costs using Medicare claims data and indirect costs (employment status and worker earnings) using regression models and nonparametric simulations. For direct costs, we estimated average spending 1 year before and after surgery. Spending estimates included physician and related services, hospital inpatient and outpatient care, and postacute care. For indirect costs, we estimated the relationship between functional status and productivity, using data from the National Health Interview Survey and regression analysis. Using regression coefficients and patient survey data, we ran a nonparametric simulation to estimate productivity (probability of working multiplied by earnings if working minus the value of missed work days) before and after THA. We used the Australian Orthopaedic Association National Joint Replacement Registry to obtain revision rates because it contained osteoarthritis-specific THA revision rates by age and gender, which were unavailable in other registry reports. Other model assumptions were extracted from a previously published cost-effectiveness analysis that included a comprehensive literature review. We incorporated all parameter estimates into Markov models to assess THA effects on quality-adjusted life years and lifetime costs. We conducted threshold and sensitivity analyses on direct costs, indirect costs, and revision rates to assess the robustness of our Markov model results. RESULTS: Compared with nonsurgical treatments, THA increased average annual productivity of patients by USD 9503 (95% CI, USD 1446-USD 17,812). We found that THA increases average lifetime direct costs by USD 30,365, which were offset by USD 63,314 in lifetime savings from increased productivity. With net societal savings of USD 32,948 per patient, total lifetime societal savings were estimated at almost USD 10 billion from more than 300,000 THAs performed in the United States each year. CONCLUSIONS: Using a Markov model approach, we show that THA produces societal benefits that can offset the costs of THA. When comparing THA with other nonsurgical treatments, policymakers should consider the long-term benefits associated with increased productivity from surgery. LEVEL OF EVIDENCE: Level III, economic and decision analysis.

Validation of bone marrow fat quantification in the presence of trabecular bone using MRI.

BACKGROUND: To validate six-echo, chemical-shift based MRI with T2 * correction for the quantification of bone marrow fat content in the presence of trabecular bone. METHODS: Ten bone phantoms were made using trabecular bone cores extracted from the distal femur and proximal tibia of 20 human cadaveric knees. Bone marrow was removed from the cores and the marrow spaces were filled with water-fat gelatin to mimic bone marrow of known fat fractions. A chemical-shift based water-fat separation method with T2 * correction was used to generate fat fraction maps. The proton density fat fractions (PDFF) between marrow regions with and without bone were compared with the reference standard of known fat fraction using the squared Pearson correlation coefficient and unpaired t-test. RESULTS: Strong correlations were found between the known fat fraction and measured PDFF in marrow without trabecular bone (R(2) = 0.99; slope = 0.99, intercept = 0.94) as well as in marrow with trabecular bone (R(2) = 0.97; slope = 1.0, intercept = -3.58). Measured PDFF between regions with and without bone were not significantly different (P = 0.5). However, PDFF was systematically underestimated by -3.2% fat fraction in regions containing trabecular bone. CONCLUSION: Our implementation of a six-echo chemical-shift based MRI pulse sequence with T2 * correction provided an accurate means of determining fat content in bone marrow in the presence of trabecular bone.

New host and lineage diversity of avian haemosporidia in the northern Andes.

The northern Andes, with their steep elevational and climate gradients, are home to an exceptional diversity of flora and fauna, particularly rich in avian species that have adapted to divergent ecological conditions. With this diversity comes the opportunity for parasites to exploit a wide breadth of avian hosts. However, little research has focused on examining the patterns of prevalence and lineage diversity of avian parasites in the Andes. Here, we screened a total of 428 birds from 19 species (representing nine families) and identified 133 infections of avian haemosporidia (31%), including lineages of Plasmodium, Haemoproteus, and Leucocytozoon. We document a higher prevalence of haemosporidia at higher elevations and lower temperatures, as well as an overall high diversity of lineages in the northern Andes, including the first sequences of haemosporidians reported in hummingbirds (31 sequences found in 11 species within the family Trochilidae). Double infections were distinguished using PHASE, which enables the separation of distinct parasite lineages. Results suggest that the ecological heterogeneity of the northern Andes that has given rise to a rich diversity of avian hosts may also be particularly conducive to parasite diversification and specialization.

Aspirin therapy in venous malformation: a retrospective cohort study of benefits, side effects, and patient experiences.

Venous malformations (VMs) are often painful and may enlarge over time. Chronic coagulopathy is common in VMs and may contribute to phleboliths and potentially to disease progression. Few studies have examined the effects of anticoagulation on VMs and to our knowledge none have examined the use of aspirin therapy. A survey was administered to patients and parents of patients with VMs who attended the University of California at San Francisco Vascular Anomalies Center over a 4-year period (2008-2012) to whom aspirin had been recommended. They were surveyed regarding whether they were taking aspirin and, if yes, whether aspirin had resulted in any appreciable benefit. Sixty-five letters were sent to potential subjects: 38 participated and 27 declined to participate or could not be contacted. Twenty-eight of the 38 had begun aspirin and 22 reported current use. Seventeen reported some benefit, including less aching (n = 2), less shooting pain (n = 15), less fullness and swelling (n = 13), and shrinking of the VM (n = 1). Discontinuation of aspirin was associated with worsening VM symptoms in five of six patients. Side effects were reported in 6 of 28 patients, including five episodes of minor bleeding or excessive bruising and one of nausea and vomiting. This study suggests that aspirin may be a beneficial treatment for VM, with a reduction in pain and soft tissue swelling and an acceptable side-effect profile, but the retrospective nature of the study and the small size of the cohort limited our conclusions. Larger prospective studies of aspirin for VM using clinical and laboratory outcome measures are needed to confirm these observations.

MI Paste Plus to prevent demineralization in orthodontic patients: a prospective randomized controlled trial.

INTRODUCTION: Enamel demineralization is a problem in orthodontics. Fluoride is partially effective in addressing this problem, but additional treatment options are needed. The objective of this prospective randomized controlled trial was to determine the effectiveness of a new product, MI Paste Plus (GC America, Alsip, Ill), in the prevention or reduction of white spot lesions in orthodontic patients. METHODS: Sixty patients who were undergoing routine orthodontic treatment were recruited for this prospective randomized clinical trial. A double-blind method of randomization was used to determine whether each patient received the MI Paste Plus or a placebo paste (Tom's of Maine, Salisbury, United Kingdom). Each patient was asked to administer the paste by using a fluoride tray for a minimum of 3 to 5 minutes each day at night after brushing. Photographic records obtained in a light-controlled environment were used to record the presence or absence of white spot lesions in both groups. The enamel decalcification index was used to determine the number of white spot lesions per surface at each time interval. Patients were followed at 4-week intervals for 3 months. A scoring system from 0 to 6 was used to determine the level of caries or cavitations. This system was also used for each tooth at each time interval. RESULTS: Fifty patients (26 using MI Paste Plus, 24 using the placebo paste) completed the study. The enamel decalcification index scores for all surfaces were 271 and 135 at the start of treatment and 126 and 258 at the end of treatment for the MI Paste Plus and placebo paste groups, respectively. The enamel decalcification index scores in the MI Paste Plus group reduced by 53.5%, whereas the placebo group increased by 91.1% during the study period. A 3-way analysis of variance (ANOVA) was done for the average enamel decalcification index scores. The surface type, the product/time interactions, and the product/surface interactions of the mean enamel decalcification index scores were significant (P <0.05). CONCLUSIONS: MI Paste Plus helped prevent the development of new white spot lesions during orthodontic treatment and decreased the number of white spot lesions already present. The placebo paste had no preventive action on white spot development during orthodontic treatment; the number of lesions actually increased. MI Paste Plus reduced white spots on the gingival surfaces; the placebo paste had the opposite effect. The incisal surface effect on the mean enamel decalcification index scores over time and between products was highly significant. The incisal enamel decalcification index scores were consistently higher than those for the other surfaces (mesial, distal, and gingival).

Evaluation of CBCT digital models and traditional models using the Little's Index.

OBJECTIVE: To determine if measurements obtained from digital models from cone beam computed tomography (CBCT) images were comparable to the traditional method of digital study models by impressions. MATERIALS AND METHODS: Digital models of 30 subjects were used. InVivoDental (Anatomage, San Jose, Calif) software was used to analyze CBCT scans taken by a Galileos cone beam scanner (Sirona, Charlotte, NC) with a field of view of 15 x 15 x 15 cm(3) and a voxel resolution of 0.125 mm. OrthoCAD (Cadent, Fairview, NJ) software was used to analyze impression scans of patients at different stages of orthodontic treatment. Impressions were taken using alginate and were mailed to OrthoCAD for digital conversion. The scans were then electronically returned in digital format for analysis. RESULTS: The maxillary mean scores for the Little's Index were 9.65 mm for digital models and 8.87 mm for InVivoDental models, respectively. The mandibular mean scores for the Little's Index were 6.41 mm for digital models and 6.27 mm for InVivoDental models, respectively. The mean overjet measurements were 3.32 mm for digital models and 3.52 mm for InVivoDental models, respectively. The overbite measurements were 2.29 mm for digital models and 2.26 mm for InVivoDental models, respectively. The paired t-test showed no statistical significance between the differences in all measurements. CONCLUSIONS: CBCT digital models are as accurate as OrthoCAD digital models in making linear measurements for overjet, overbite, and crowding measurements.

Analysis of CD25hiCD4+ "regulatory" T-cell subtypes in atopic dermatitis reveals a novel T(H)2-like population.

BACKGROUND: It is unresolved whether circulating CD25hiCD4+ T cells in patients with atopic dermatitis who have elevated IgE (IgE(high)) are regulatory or effector in nature. OBJECTIVE: To analyze the properties of CD25hi T-cell subtypes in IgE(high) atopic dermatitis. METHODS: The phenotype of circulating CD25hi T cells was analyzed by flow cytometry using PBMCs from patients with atopic dermatitis (total IgE > 250 IU/mL). Cytokines induced in CD25hi subtypes were analyzed after activation with anti-CD3 mAb (+/-IL-2) and in the presence of activated autologous effector T cells (CD25negCD4+). Reactivity to bacterial superantigen derived from the skin-colonizing organism Staphylococcus aureus was also evaluated. RESULTS: CD25(hi) T cells expressing regulatory T-cell markers (Foxp3, CCR4, cutaneous lymphocyte-associated antigen) were increased in atopic dermatitis compared with IgE(low) controls. This phenomenon was linked to disease severity. Two subtypes of CD25hi T cells were identified on the basis of differential expression of the chemokine receptor CCR6. Although the ratio of CCR6+ and CCR6neg subtypes within the CD25hi subset was unaltered in atopic dermatitis, each subtype proliferated spontaneously ex vivo, suggesting in vivo activation. Activated CCR6neg cells secreted T(H)2 cytokines, and coculture with effector T cells selectively enhanced IL-5 production. Moreover, induction of a T(H)2-dominated cytokine profile on activation with bacterial superantigen was restricted to the CCR6neg subtype. CONCLUSION: Despite a regulatory phenotype, activated CD25hi T cells that lack expression of CCR6 promote T(H)2 responses.