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1.  Antigen-specific TGF-β-induced regulatory T cells but not natural Tregs ameliorate autoimmune arthritis by shifting the balance of Th17 toward Treg cells 
Arthritis and rheumatism  2012;64(8):2548-2558.
Transferred CD4+CD25+Foxp3+ regulatory cells (Tregs) can prevent autoimmune disease, but generally fail to ameliorate established disease. Here we demonstrate that antigen-specific Tregs induced with IL-2 and TGF-β ex-vivo (iTregs), but not equivalent expanded thymus-derived nTregs can prevent progression of established collagen-induced arthritis.
DBA/1 mice with collagen-induced arthritis were treated with iTreg or nTreg generated or expanded in vitro before or shortly after collagen II immunization, and clinical scores were determined. Inflammatory responses were determined by measuring the levels of anti-CII antibody in the serum and histological pathology in joints. The Th1/Th17-mediated autoreactive response was evaluated by determining the cytokine profile of draining lymph node cells by flow cytometry.
Following transfer, nTregs exhibited decreased Foxp3 and Bcl-2 expression, decreased suppressive activity, and many converted to Th17 cells. By contrast, transferred iTregs were more numerous, retained Foxp3 expression and their suppressive activity in the presence of IL-6, and were resistant to Th17 conversion. Remarkably, ten days after transfer of donor iTregs shifted the predominance from Th17 to Treg cells in recipient draining LNs.
These findings provide evidence that transferred TGF-β-induced iTregs are more stable and functional than nTregs in mice with established autoimmunity. Moreover, iTregs can have tolerogenic effects even in the presence of ongoing inflammation. The therapeutic potential of human iTregs in subjects with chronic, immune-mediated inflammatory diseases deserves to be investigated.
PMCID: PMC4364395  PMID: 22605463
2.  Therapeutic Polyclonal human CD8+ Fox3+ TNFR2+ PD-L1+ Regulatory Cells Induced ex-vivo 
Clinical immunology (Orlando, Fla.)  2013;149(3):450-463.
We report that polyclonal CD8regs generated in one week ex-vivo with anti-CD3/28 beads and cytokines rapidly developed suppressive activity in vitro sustained by TGF-β. In immunodeficient mice, these CD8regs demonstrated a markedly protective, IL-10 dependent activity against a xeno-GVHD. They expressed IL-2Rα/β, Foxp3, TNFR2, and the negative co-stimulatory receptors CTLA-4, PD-1, PD-L1 and Tim-3. Suppressive activity in vitro correlated better with TNFR2 and PD-L1 than Foxp3. Blocking studies suggested that TNF enhanced PD-L1 expression and the suppressive activity of the CD8regs generated. Unlike other polyclonal CD4 and CD8 Tregs, these CD8regs preferentially targeted allogeneic T cells, but they lacked cytotoxic activity against them even after sensitization. Unlike CD4regs, these CD8regs could produce IL-2 and proliferate while inhibiting target cells. If these CD8regs can persist in foreign hosts without impairing immune surveillance, they could serve as a practical remission-inducing product for the treatment of autoimmune diseases, graft-versus-host disease, and allograft rejection.
PMCID: PMC3941976  PMID: 24211847
3.  Polyclonal CD4+Foxp3+ Treg cells induce TGFβ-dependent tolerogenic dendritic cells that suppress the murine lupus-like syndrome 
Interplay between Foxp3+ regulatory T cells (Treg) and dendritic cells (DCs) maintains immunologic tolerance, but the effects of each cell on the other are not well understood. We report that polyclonal CD4+Foxp3+ Treg cells induced ex vivo with transforming growth factor beta (TGFβ) (iTreg) suppress a lupus-like chronic graft-versus-host disease by preventing the expansion of immunogenic DCs and inducing protective DCs that generate additional recipient CD4+Foxp3+ cells. The protective effects of the transferred iTreg cells required both interleukin (IL)-10 and TGFβ, but the tolerogenic effects of the iTreg on DCs, and the immunosuppressive effects of these DCs were exclusively TGFβ-dependent. The iTreg were unable to tolerize Tgfbr2-deficient DCs. These results support the essential role of DCs in ‘infectious tolerance’ and emphasize the central role of TGFβ in protective iTreg/DC interactions in vivo.
PMCID: PMC3523557  PMID: 22773728
regulatory T cells; dendritic cells; TGFβ; graft-versus-host disease
4.  Cutting Edge: All-Trans Retinoic Acid Sustains the Stability and Function of Natural Regulatory T Cells in an Inflammatory Milieu 
Recent studies have demonstrated that plasticity of naturally occurring CD4+Foxp3+ regulatory T cells (nTregs) may account for their inability to control chronic inflammation in established autoimmune diseases. All-trans retinoic acid (atRA), the active derivative of vitamin A, has been demonstrated to promote Foxp3+ Treg differentiation and suppress Th17 development. In this study, we report a vital role of atRA in sustaining the stability and functionality of nTregs in the presence of IL-6. We found that nTregs treated with atRA were resistant to Th17 and other Th cell conversion and maintained Foxp3 expression and suppressive activity in the presence of IL-6 in vitro. atRA decreased IL-6R expression and signaling by nTregs. Of interest, adoptive transfer of nTregs even from arthritic mice treated with atRA suppressed progression of established collagen-induced arthritis. We suggest that nTregs treated with atRA may represent a novel treatment strategy to control established chronic immune-mediated inflammatory diseases.
PMCID: PMC3098624  PMID: 20679534
5.  Role of SMAD and Non-SMAD Signals in the Development of Th17 and Regulatory T Cells 
Whereas TGF-β is essential for the development of peripherally induced Foxp3+ regulatory T cells (iTreg cells) and Th17 cells, the intracellular signaling mechanism by which TGF-β regulates development of both cell subsets is less understood. In this study, we report that neither Smad2 nor Smad3 gene deficiency abrogates TGF-β–dependent iTreg induction by a deacetylase inhibitor trichostatin A in vivo, although the loss of the Smad2 or Smad3 gene partially reduces iTreg induction in vitro. Similarly, SMAD2 and SMAD3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis. In addition, ERK and/or JNK pathways were shown to be involved in regulating iTreg cells, whereas the p38 pathway predominately modulated Th17 and experimental autoimmune encephalomyelitis induction. Therefore, selective targeting of these intracellular TGF-β signaling pathways during iTreg and Th17 cell development might lead to the development of therapies in treating autoimmune and other chronic inflammatory diseases.
PMCID: PMC3087811  PMID: 20304828
6.  Correction: Characterization of Protective Human CD4+CD25+ FOXP3+ Regulatory T Cells Generated with IL-2, TGF-β and Retinoic Acid 
PLoS ONE  2011;6(1):10.1371/annotation/8f29ac57-8219-4ffd-84c9-5e6bf6dcd017.
PMCID: PMC3021482
7.  Synergistic effect of TGF-β superfamily members on the induction of Foxp3+ regulatory T cells1 
European journal of immunology  2010;40(1):142-152.
TGF-β plays an important role in the induction of regulatory T cells (Treg) and maintenance of immunologic tolerance, but whether other members of TGF-β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF-β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP-2 and BMP-4, are unable to induce non-regulatory T cells to become Foxp3+ Treg. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-β to induce naive CD4+CD25- cells to express Foxp3; however, BMP-2/4 and TGF-β have a synergistic effect on the induction of Foxp3+ Treg. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3+ Treg induced by TGF-β. Data further advocate that TGF-β is a key signaling factor for Foxp3+ Treg development. In addition, the synergistic effect of BMP-2/4 and TGF-β indicates that the simultaneous manipulation of TGF-β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.
PMCID: PMC2837277  PMID: 19943263
BMP; TGF-β; Foxp3; regulatory T cells
8.  Diabetes Interactive Diary: A New Telemedicine System Enabling Flexible Diet and Insulin Therapy While Improving Quality of Life 
Diabetes Care  2009;33(1):109-115.
Widespread use of carbohydrate counting is limited by its complex education. In this study we compared a Diabetes Interactive Diary (DID) with standard carbohydrate counting in terms of metabolic and weight control, time required for education, quality of life, and treatment satisfaction.
Adults with type 1 diabetes were randomly assigned to DID (group A, n = 67) or standard education (group B, n = 63) and followed for 6 months. A subgroup also completed the SF-36 Health Survey (SF-36) and World Health Organization-Diabetes Treatment Satisfaction Questionnaire (WHO-DTSQ) at each visit.
Of 130 patients (aged 35.7 ± 9.4 years; diabetes duration 16.5 ± 10.5 years), 11 dropped out. Time for education was 6 h (range 2–15 h) in group A and 12 h (2.5–25 h) in group B (P = 0.07). A1C reduction was similar in both groups (group A from 8.2 ± 0.8 to 7.8 ± 0.8% and group B from 8.4 ± 0.7 to 7.9 ± 1.1%; P = 0.68). Nonsignificant differences in favor of group A were documented for fasting blood glucose and body weight. No severe hypoglycemic episode occurred. WHO-DTSQ scores increased significantly more in group A (from 26.7 ± 4.4 to 30.3 ± 4.5) than in group B (from 27.5 ± 4.8 to 28.6 ± 5.1) (P = 0.04). Role Physical, General Health, Vitality, and Role Emotional SF-36 scores improved significantly more in group A than in group B.
DID is at least as effective as traditional carbohydrate counting education, allowing dietary freedom for a larger proportion of type 1 diabetic patients. DID is safe, requires less time for education, and is associated with lower weight gain. DID significantly improved treatment satisfaction and several quality-of-life dimensions.
PMCID: PMC2797954  PMID: 19808926
9.  Characterization of Protective Human CD4+CD25+ FOXP3+ Regulatory T Cells Generated with IL-2, TGF-β and Retinoic Acid 
PLoS ONE  2010;5(12):e15150.
Protective CD4+CD25+ regulatory T cells bearing the Forkhead Foxp3 transcription factor can now be divided into three subsets: Endogenous thymus-derived cells, those induced in the periphery, and another subset induced ex-vivo with pharmacological amounts of IL-2 and TGF-β. Unfortunately, endogenous CD4+CD25+ regulatory T cells are unstable and can be converted to effector cells by pro-inflammatory cytokines. Although protective Foxp3+CD4+CD25+ cells resistant to proinflammatory cytokines have been generated in mice, in humans this result has been elusive. Our objective, therefore, was to induce human naïve CD4+ cells to become stable, functional CD25+ Foxp3+ regulatory cells that were also resistant to the inhibitory effects of proinflammatory cytokines.
Methodology/Principal Findings
The addition of the vitamin A metabolite, all-trans retinoic acid (atRA) to human naïve CD4+ cells suboptimally activated with IL-2 and TGF-β enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of naïve to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-β enabled CD4+CD45RA+ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-β-induced CD4+ regs were anergic and low producers of IL-2. They had potent in vitro suppressive activity and protected immunodeficient mice from a human-anti-mouse GVHD as well as expanded endogenous Tregs. However, treatment of endogenous Tregs with IL-1β and IL-6 decreased FOXP3 expression and diminished their protective effects in vivo while atRA-induced iTregs were resistant to these inhibitory effects.
We have developed a methodology that induces human CD4+ cells to rapidly become stable, fully functional suppressor cells that are also resistant to proinflammatory cytokines. This methodology offers a practical novel strategy to treat human autoimmune diseases and prevent allograft rejection without the use of agents that kill cells or interfere with signaling pathways.
PMCID: PMC3003689  PMID: 21179414
10.  Identity of mysterious CD4+CD25-Foxp3+ cells in SLE 
Various abnormalities in CD4+CD25+ regulatory T cells (Tregs) in systemic lupus erythematosus (SLE) include increased Foxp3+ cells that are CD25 negative. Barring methodological technical factors, these cells could be atypical Tregs or activated non-Treg CD4+ cells that express Foxp3. Two groups have reached opposite conclusions that could possibly reflect the subjects studied. One group studied untreated new-onset SLE and suggested that these T cells were mostly CD25-Foxp3+ non-Tregs. The other group studied patients with long-standing disease and suggested that these cells are mostly dysfunctional Tregs. A third group reported increased Foxp3+CD4+CD25dim rather than CD25- cells in active SLE and these were also non-Tregs. Thus, it is likely that not all Foxp3+ T cells in SLE have protective suppressive activity.
PMCID: PMC2875622  PMID: 20122288
11.  Regulatory T cells in systemic lupus erythematosus: past, present and future 
Regulatory/suppressor T cells (Tregs) maintain immunologic homeo-stasis and prevent autoimmunity. In this article, past studies and recent studies of Tregs in mouse models for lupus and of human systemic lupus erythematosus are reviewed concentrating on CD4+CD25+Foxp3+ Tregs. These cells consist of thymus-derived, natural Tregs and peripherally induced Tregs that are similar phenotypically and functionally. These Tregs are decreased in young lupus-prone mice, but are present in normal numbers in mice with established disease. In humans, most workers report CD4+Tregs are decreased in subjects with active systemic lupus erythematosus, but the cells increase with treatment and clinical improvement. The role of immunogenic and tolerogenic dendritic cells in controlling Tregs is discussed, along with new strategies to normalize Treg function in systemic lupus erythematosus.
PMCID: PMC2656253  PMID: 19040771
12.  Accuracy Requirements for a Hypoglycemia Detector: An Analytical Model to Evaluate the Effects of Bias, Precision, and Rate of Glucose Change 
There has been considerable debate on what constitutes a good hypoglycemia (Hypo) detector and what is the accuracy required from the continuous monitoring sensor to meet the requirements of such a detector. The performance of most continuous monitoring sensors today is characterized by the mean absolute relative difference (MARD), whereas Hypo detectors are characterized by the number of false positive and false negative alarms, which are more relevant to the performance of a Hypo detector. This article shows that the overall accuracy of the system and not just the sensor plays a key role.
A mathematical model has been developed to investigate the relationship between the accuracy of the continuous monitoring system as described by the MARD, and the number of false negatives and false positives as a function of blood glucose rate change is established. A simulation method with N = 10,000 patients is used in developing the model and generating the results.
Based on simulation for different scenarios for rate of change (0.5, 1.0, and 5.0 mg/dl per minute), sampling rate (from 1, 2.5, 5, and 10 minutes), and MARD (5, 7.5, 10, 12.5, and 15%), the false positive and false negative ratios are computed. The following key results are from these computations.
1. For a given glucose rate of change, there is an optimum sampling time.
2. The optimum sampling time as defined in the critical sampling rate section gives the best combination of low false positives and low false negatives.
3. There is a strong correlation between MARD and false positives and false negatives.
4. For false positives of <10% and false negatives of <5%, a MARD of <7.5% is needed.
Based on the model, assumptions in the model, and the simulation on N = 10,000 patients for different scenarios for rate of glucose change, sampling rate, and MARD, it is concluded that the false negative and false positive ratio will vary depending on the alarm Hypo threshold set by the patient and the MARD value. Also, to achieve a false negative ratio <5% and a false positive ratio <10% would require continuous glucose monitoring to have an MARD ≤7.5%.
PMCID: PMC2769658  PMID: 19885133
accuracy; alarm threshold; bias; calibration; critical sampling rate; coefficient of variation; continuous glucose monitor; critical threshold; drift; false positive ratio; false negative ratio; glucose monitoring system; hypoglycemia; hypoglycemia detector; hypoglycemia threshold; lag effect; linear regression; mean absolute relative difference; precision; random variation; rate of glucose change; relative bias; sampling rate; sampling time; slope; standard normal random variable; systematic bias; target area; variable sampling rate
13.  The potential of human regulatory T cells generated ex vivo as a treatment for lupus and other chronic inflammatory diseases 
Arthritis Research  2002;4(4):241-246.
Regulatory T cells prevent autoimmunity by suppressing the reactivity of potentially aggressive self-reactive T cells. Contact-dependent CD4+ CD25+ 'professional' suppressor cells and other cytokine-producing CD4+ and CD8+ T-cell subsets mediate this protective function. Evidence will be reviewed that T cells primed with transforming growth factor (TGF)-β expand rapidly following restimulation. Certain CD4+ T cells become contact-dependent suppressor cells and other CD4+ and CD8+ cells become cytokine-producing regulatory cells. This effect is dependent upon a sufficient amount of IL-2 in the microenvironment to overcome the suppressive effects of TGF-β. The adoptive transfer of these suppressor cells generated ex vivo can protect mice from developing chronic graft-versus-host disease with a lupus-like syndrome and alter the course of established disease. These data suggest that autologous T cells primed and expanded with TGF-β have the potential to be used as a therapy for patients with systemic lupus erythematosus and other chronic inflammatory diseases. This novel adoptive immunotherapy also has the potential to prevent the rejection of allogeneic transplants.
PMCID: PMC128930  PMID: 12106494
autoimmunity; IL-2; regulatory T cells; systemic lupus erythematosus; transforming growth factor-β
14.  Potency of Synthroid Tablets 
Western Journal of Medicine  1984;141(4):525.
PMCID: PMC1021881  PMID: 18749650
The Journal of Experimental Medicine  1972;136(6):1631-1647.
Tumor immunity in patients with primary intracranial tumors was assessed in relation to the general status of host immunocompetence. Lymphocyte sensitization to tumor-specific membrane antigens was demonstrated by the proliferative response of lymphocytes in the presence of autochthonous tumor cells. Paradoxically, one-half of the patients could not be sensitized to a primary antigen, dinitrochlorobenzene; existing delayed hypersensitivity was also depressed, as measured by skin tests and lymphocyte transformation in response to common antigens. A heat-stable factor in patients' sera blocked cell-mediated tumor immunity. In addition, these "enhancing" sera consistently suppressed the blastogenic response of autologous and homologous lymphocytes to phytohemagglutinin and to membrane antigens on allogeneic cells in the one-way mixed lymphocyte culture. When patients' leukocytes were washed and autologous plasma replaced with normal plasma, reactivity in the mixed lymphocyte culture increased to normal values. In vitro immunosuppressive activity in patients' plasma or sera correlated with depressed delayed hypersensitivity. After removal of the tumor, suppressor activity disappeared. IgG fractions of patient sera contained strong immunosuppressive activity. These data suggest that the suppressor factor may be an isoantibody elicited by the tumor that also binds to receptors on the lymphocyte membrane. In addition to specifically blocking cell-mediated tumor immunity, enhancing sera may broadly depress host immunocompetence.
PMCID: PMC2139329  PMID: 4345108
16.  The Development of Macrophages from Large Mononuclear Cells in the Blood of Patients with Inflammatory Disease 
Journal of Clinical Investigation  1972;51(4):760-768.
The origin and function of the increased of “atypical lymphocytes” which appear in the blood of patients with many inflammatory diseases is not known. Leukocyte suspensions from eight patients with systemic lupus erythematosus (SLE), five patients with other rheumatic diseases, and five patients with infectious diseases were pulse-labeled with tritiated thymidine (Tdr-3H) and sampled after 5 and 72 hr in vitro. Radioautographs indicated that 35% of the total large, nonphagocytic mononuclear leukocytes incorporated Tdr-3H during the initial 5 hr of culture. Tdr-3H-labeled large phagocytic or glass-adherent cells were observed only infrequently. After 72 hr one-third of the original number of Tdr-3H-labeled cells from patients with SLE developed the morphology of macrophages and the capacity to phagocytose latex particles. Similar findings were observed in patients with other rheumatic diseases and bacterial infections. In contrast, the thymidine-labeled cells from patients with infectious hepatitis and infectious mononucleosis were poorly viable in culture and rarely became macrophages. Tdr-3H-labeled small lymphocytes were uncommon. The present experiments suggest that in patients with certain inflammatory diseases large, proliferating “lymphocytelike” cells are very immature monocyte precursors which appear in response to tissue injury. These DNA-synthesizing cells together with mature monocytes may serve as the circulating source of macrophages.
PMCID: PMC302188  PMID: 5014610
17.  Biochemical and pharmacologic effects of α-methyltyrosine in man 
Journal of Clinical Investigation  1968;47(3):577-594.
Alpha methyltyrosine (α-MPT) was administered to 52 patients from 4 days to 10 months; 22 patients were cases of pheochromocytoma and 20 had essential hypertension. Inhibition of catecholamine synthesis in the range of 50-80% was achieved with divided daily drug dosage of from 1.0 to 4.0 g. Striking clinical benefit was noted in patients with pheochromocytoma in whom the drug was used in preparation for surgery and during chronic medical management. The drug appeared to have limited usefulness when used in essential hypertension, unless added to existing therapy with conventional agents. No beneficial effects were noted in thyrotoxicosis, glaucoma, and Raynaud's phenomenon. Untoward effects in order of decreasing incidence were: sedation (with insomnia on withdrawal), anxiety, tremor, diarrhea, and galactorrhea. Drug crystalluria, which has been observed in animals and is currently restrictive of clinical trials, was not observed in these studies. Evidence is presented that the minor conversion of α-MPT to methyldopa probably does not contribute significantly to the central and peripheral effects of the drug.
PMCID: PMC297204  PMID: 5637145
18.  Lactation Due to Methyldopa 
British Medical Journal  1963;1(5343):1460.
PMCID: PMC2124093  PMID: 13942986

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