Low TCR signal strength induces combined expansion of Th2 and regulatory T cell populations that protect mice from the development of type 1 diabetes.

AIMS/HYPOTHESIS: Weak stimulation of CD4(+) T cells induces expansion of CD4(+) forkhead box P3(+) regulatory T cells (Tregs) and can also promote T helper (Th) 2 responses, which have demonstrable beneficial effects on autoimmune diabetes. This study explored the feasibility of combined Treg/Th2 expansion for immunotherapy of type 1 diabetes in NOD mice. METHODS: We compared Treg and Th responses to dendritic cells (DC) presenting scaled antigen doses to islet-specific NOD CD4(+) T cells. Flow cytometric and Luminex analyses were performed to determine the phenotype and cytokine profile of expanded T cells. The ability of expanded T cells to prevent type 1 diabetes was tested in an adoptive transfer model. RESULTS: In vitro studies revealed a hierarchical, selective expansion of Treg and T effector (Teff) populations at different antigen doses. Thus, a single low dose produced a mixture of Tregs Th2 and type 1 regulatory (Tr1) cells, which prevented diabetes in NOD-SCID mice and increased the ratio of Treg/Teff cells infiltrating the pancreatic islets. Subcutaneous injection of DC, previously shown to prevent diabetes in NOD mice, induced expansion of the same mixture of Tregs Tr1 and Th2 cells. Low-dose expansion of Treg required MHC-T cell receptor interaction and was partly dependent on T cell derived TGF-ß and IL-2. Autocrine IFN-γ was required for the promotion of diabetogenic Th1 cells at high antigen doses. CONCLUSIONS/INTERPRETATION: Weak stimulation of CD4(+) T cells with DC and low-dose antigen expands a combination of antigen-specific Tregs Th2 and Tr1 cells that prevent autoimmunity, without the need to target or purify specific Treg populations.

Increased levels of invariant natural killer T lymphocytes worsen metabolic abnormalities and atherosclerosis in obese mice.

Obesity is a chronic inflammatory state characterized by infiltration of adipose tissue by immune cell populations, including T lymphocytes. Natural killer T (NKT) cells, a specialized lymphocyte subset recognizing lipid antigens, can be pro- or anti-inflammatory. Their role in adipose inflammation continues to be inconclusive and contradictory. In obesity, the infiltration of tissues by invariant NKT (iNKT) cells is decreased. We therefore hypothesized that an excess iNKT cell complement might improve metabolic abnormalities in obesity. Vα14 transgenic (Vα14tg) mice, with increased iNKT cell numbers, on a LDL receptor-deficient (Ldlr(-/-)) background and control Ldlr(-/-) mice were placed on an obesogenic diet for 16 weeks. Vα14tg.Ldlr(-/-) mice gained 25% more weight and had increased adiposity than littermate controls. Transgenic mice also developed greater dyslipidemia, hyperinsulinemia, insulin resistance, and hepatic triglyceride accumulation. Increased macrophage Mac2 immunostaining and proinflammatory macrophage gene expression suggested worsened adipose inflammation. Concurrently, these mice had increased atherosclerotic lesion area and aortic inflammation. Thus, increasing the complement of iNKT cells surprisingly exacerbated the metabolic, inflammatory, and atherosclerotic features of obesity. These findings suggest that the reduction of iNKT cells normally observed in obesity may represent a physiological attempt to compensate for this inflammatory condition.

Extremely large telescopes at risk.

Images of the cosmos from the Hubble Space Telescope and the James Webb Space Telescope have awed the public and astronomers alike. Until the Hubble, breakthroughs in astronomy came from big telescopes on mountain-top observatories-discoveries that include the expansion of the Universe and planets orbiting other stars. A new generation of extremely large ground-based telescopes is under development, which, when paired with space-based observatories, will produce even more remarkable discoveries about our Universe.

Prostaglandin E2-induced synaptic plasticity in neocortical networks of organotypic slice cultures.

Traumatic brain injury (TBI) is a major cause of epilepsy, yet the mechanisms underlying the progression from TBI to epilepsy are unknown. TBI induces the expression of COX-2 (cyclooxygenase-2) and increases levels of prostaglandin E2 (PGE2). Here, we demonstrate that acutely applied PGE2 (2 mum) decreases neocortical network activity by postsynaptically reducing excitatory synaptic transmission in acute and organotypic neocortical slices of mice. In contrast, long-term exposure to PGE2 (2 mum; 48 h) presynaptically increases excitatory synaptic transmission, leading to a hyperexcitable network state that is characterized by the generation of paroxysmal depolarization shifts (PDSs). PDSs were also evoked as a result of depriving organotypic slices of activity by treating them with tetrodotoxin (TTX, 1 mum; 48 h). This treatment predominantly increased postsynaptically excitatory synaptic transmission. The network and cellular effects of PGE2 and TTX treatments reversed within 1 week. Differences in the underlying mechanisms (presynaptic vs postsynaptic) as well as occlusion experiments in which slices were exposed to TTX plus PGE2 suggest that the two substances evoke distinct forms of homeostatic plasticity, both of which result in a hyperexcitable network state. PGE2 and TTX (alone or together with PGE2) also increased levels of apoptotic cell death in organotypic slices. Thus, we hypothesize that the increase in excitability and apoptosis may constitute the first steps in a cascade of events that eventually lead to epileptogenesis triggered by TBI.

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion.

The definitions of tolerogenic vs immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allogeneic Ags. However, we have previously reported that mature DC (mDC) prevented the onset of autoimmune diabetes, whereas immature DC (iDC) were therapeutically ineffective. In this study, islet-specific CD4(+) T cells from BDC2.5 TCR-transgenic mice were stimulated in the absence of exogenous cytokine with iDC or mDC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both iDC and mDC presenting low peptide doses induced weak TCR signaling via the Akt/mammalian target of rapamycin (mTOR) pathway, resulting in significant expansion of Foxp3(+) Treg. Furthermore, unpulsed mDC, but not iDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3(neg) Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-transgenic T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with Ag dose and inversely with Treg expansion. Studies with T cells or DC from IL-6(-/-) mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low Ag doses. These studies indicate that the strength of Akt/mTOR signaling, a critical T cell-intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production.

Medical practice perspective: identification and mitigation of risk factors for mortality associated with intrathecal opioids for non-cancer pain.

OBJECTIVE: The authors recently determined that early and longer term mortality after initiation or reinitiation of intrathecal opioid therapy is higher than previously appreciated: 0.088% within 3 days, 0.39% at 1 month, and 3.89% at 1 year. These rates were 7.5 (confidence interval, 5.7-9.8), 3.4 (confidence interval, 2.9-3.8), and 2.7 (confidence interval, 2.6-2.8) times higher, respectively, at each interval than expected based on the age- and gender-matched general U.S. population. A substantial portion of this excess mortality is probably therapy related and cannot be entirely accounted for by underlying demographic or patient-related factors, or by device malfunctions. We also analyzed multiple complementary internal, governmental, and insurance databases to quantify mortality and to identify medical practice patterns that appear to be associated with patient mortality risks, and to suggest measures for physicians and health care facilities to consider in order to reduce those risks. Both of those objectives involve judgments, which may be controversial and are subject to practical limitations. RESULTS: Multiple clinical and patient- or therapy-related factors appear to increase the risk for early post-implant mortality. Specific risk mitigation measures associated with each factor include: close attention to the starting intrathecal opioid dose (or restarting dose after therapy interruption); avoidance of outpatient implant or other device procedures that involve less than 24-hour monitoring for respiratory depression; supervision of concomitant opioid, respiratory depressant, or other central nervous system active drug intake early post-implant and chronically in the outpatient setting; and careful programming or dosage calculations and decisions in order to avoid the unintentional administration of high intrathecal opioid drug doses. CONCLUSIONS: Mortality after initiation of or device interventions in intrathecal drug delivery patients appears to occur as a result of multiple factors that present possible mitigation opportunities for physicians and health care facilities.

In vivo cytometry of antigen-specific t cells using 19F MRI.

Noninvasive methods to image the trafficking of phenotypically defined immune cells are paramount as we attempt to understand adaptive immunity. A (19)F MRI-based methodology for tracking and quantifying cells of a defined phenotype is presented. These methods were applied to a murine inflammation model using antigen-specific T cells. The T cells that were intracellularly labeled ex vivo with a perfluoropolyether (PFPE) nanoemulsion and cells were transferred to a host receiving a localized inoculation of antigen. Longitudinal (19)F MRI over 21 days revealed a dynamic accumulation and clearance of T cells in the lymph node (LN) draining the antigen. The apparent T-cell numbers were calculated in the LN from the time-lapse (19)F MRI data. The effect of in vivo T-cell division on the (19)F MRI cell quantification accuracy was investigated using fluorescence assays. Overall, in vivo cytometry using PFPE labeling and (19)F MRI is broadly applicable to studies of whole-body cell biodistribution.

Mortality associated with implantation and management of intrathecal opioid drug infusion systems to treat noncancer pain.

BACKGROUND: In 2006, the authors observed a cluster of three deaths, which circumstances suggested were opioid-related, within 1 day after placement of intrathecal opioid pumps for noncancer pain. Further investigation suggested that mortality among such patients was higher than previously appreciated. The authors performed investigations to quantify that mortality and compare the results to control populations, including spinal cord stimulation and low back surgery. METHODS: After analyzing nine index cases--three sentinel cases and six identified by a prospective strategy--the authors used epidemiological methods to investigate whether mortality rates reflected patient- or therapy-related differences. Mortality rates after intrathecal opioid therapy and spinal cord stimulation were derived by correlating Medtronic device registration data with de-identified data from the Social Security Death Master File. Aggregate demographic and comorbidity data were obtained from Medicare and United Healthcare population databases to examine the influence of demographics and comorbidities on mortality. RESULTS: Device registration and Social Security analyses revealed an intrathecal opioid therapy mortality rate of 0.088% at 3 days after implantation, 0.39% at 1 month, and 3.89% at 1 yr-a higher mortality than after spinal cord stimulation implants or after lumbar diskectomy in community hospitals. Demographic, illness profile, and mortality analyses of large databases suggest, despite limitations, that excess mortality was related to intrathecal opioid therapy, and could not be fully explained by other factors. These findings were consistent with the nine index cases that revealed that respiratory arrest caused or contributed to death in all patients. No device malfunctions associated with overinfusion were identified among cases where data were available. CONCLUSIONS: Patients with noncancer pain treated with intrathecal opioid therapy experience increased mortality compared to similar patients treated by using other therapies. Respiratory depression as a consequence of intrathecal drug overdosage or mixed intrathecal and systemic drug interactions is one plausible, but hypothetical mechanism. The exact causes for patient deaths and the proportion of those deaths attributable to intrathecal opioid therapy remain to be determined. These findings, although based on incomplete information, suggest that it may be possible to reduce mortality in noncancer intrathecal opioid therapy patients.

Naturally occurring regulatory T cells: recent insights in health and disease.

Development of naturally occurring CD4+CD25+ T regulatory cells (Treg) in the thymus requires the transcription factor Foxp3. Major histocompatibility complex (MHC) class II, self-ligands expressed by epithelial cells, and thymic stromal lymphopoietin also appear to play important roles. In addition, several molecular regulators of T-cell receptor (TCR) signaling (both positive and negative) have been implicated in the control of Treg development. Foxp3 is a transcriptional repressor of IL-2 and other cytokines and appears to maintain the anergic and suppressor function of these cells. Multiple cell types (T cells, B cells, dendritic cells [DC], and natural killer [NK] cells) are targeted by Treg using diverse suppressor mechanisms, whereas factors that regulate Treg proliferation and function, including Toll-like receptor (TLR) ligands, have also been identified. Because Treg play an important role in the control of autoimmunity, therapeutic strategies are being pursued to enhance their numbers and function in specific autoimmune diseases. In transplantation, where Treg also offer potential for therapy of rejection and graft-versus-host disease (GVHD), indirect allorecognition may be the dominant pathway for immune regulation by these cells. In tumor immunology, Treg have emerged as major suppressors of T-cell-mediated antitumor responses and represent a significant obstacle to effective anticancer vaccines. Strategies aimed at depletion/functional inhibition of these cells by molecular targeting must maintain a critical balance between tumor immunity and self-tolerance.

Mucins in gastrointestinal cancers.

The mucin family has been under study by molecular biologists, biochemists, pathologists and immunologists interested in cancer because of the role these molecules can play in the diagnosis and treatment of cancer. Immense knowledge has been accumulated, but the high speed of progress in the laboratory has not been matched by the progress towards applying this knowledge in the clinic. For example, specific knowledge of cancer-associated changes in the expression and glycosylation of various mucins, which can aid in the diagnosis as well as prognosis of GI cancers, has not yet led to the use of a panel of anti-mucin antibodies as a standard diagnostic tool. Similarly, many more opportunities exist for using mucin-based therapies than are currently being considered in the clinic. This chapter aimed to highlight some of these opportunities and to interest clinician scientists in exploring them in the near future.

Intrathecal baclofen withdrawal mimicking sepsis.

Baclofen (Lioresal) is a drug of choice to treat spasticity and is increasingly being administered intrathecally via an implantable pump in cases refractory to oral therapy. Emergency physicians will likely treat patients with baclofen withdrawal or overdose as this treatment becomes more widespread. The syndrome of baclofen withdrawal presents with altered mental status, fever, tachycardia, hypertension or hypotension, seizures, and rebound spasticity, and may be fatal if not treated appropriately. Baclofen withdrawal may mimic other diseases including sepsis, meningitis, autonomic dysreflexia, malignant hyperthermia, or neuroleptic malignant syndrome. Treatment consists of supportive care, reinstitution of baclofen, benzodiazepines, and diagnosis and eventual repair of intrathecal pump and catheter malfunction.

The duration of T cell stimulation is a critical determinant of cell fate and plasticity.

Variations in T cell receptor (TCR) signal strength, as indicated by differential activation of downstream signaling pathways, determine the fate of naïve T cells after encounter with antigen. Low-strength signals favor differentiation into regulatory T (T(reg)) cells containing the transcription factor Foxp3, whereas high-strength signals favor generation of interleukin-2-producing T helper (T(H)) cells. We constructed a logic circuit model of TCR signaling pathways, a major feature of which is an incoherent feed-forward loop involving both TCR-dependent activation of Foxp3 and its inhibition by mammalian target of rapamycin (mTOR), leading to the transient appearance of Foxp3(+) cells under T(H) cell-generating conditions. Experiments confirmed this behavior and the prediction that the immunosuppressive cytokine TGF-ß (transforming growth factor-ß) could generate T(reg) cells even during continued Akt-mTOR signaling. We predicted that sustained mTOR activity could suppress FOXP3 expression upon TGF-ß removal, suggesting a possible mechanism for the experimentally observed instability of Foxp3(+) cells. Our model predicted, and experiments confirmed, that transient stimulation of cells with high-dose antigen generated T(H), T(reg), and nonactivated cells in proportions depending on the duration of TCR stimulation. Experimental analysis of cells after antigen removal identified three populations that correlated with these T cell fates. Further analysis of simulations implicated a negative feedback loop involving Foxp3, the phosphatase PTEN, and Akt-mTOR in determining fate. These results suggest that there is a critical time after TCR stimulation during which heterogeneity in the differentiating population of cells leads to increased plasticity of cell fate.

T cell activation inhibitors reduce CD8+ T cell and pro-inflammatory macrophage accumulation in adipose tissue of obese mice.

Adipose tissue inflammation and specifically, pro-inflammatory macrophages are believed to contribute to insulin resistance (IR) in obesity in humans and animal models. Recent studies have invoked T cells in the recruitment of pro-inflammatory macrophages and the development of IR. To test the role of the T cell response in adipose tissue of mice fed an obesogenic diet, we used two agents (CTLA-4 Ig and anti-CD40L antibody) that block co-stimulation, which is essential for full T cell activation. C57BL/6 mice were fed an obesogenic diet for 16 weeks, and concomitantly either treated with CTLA-4 Ig, anti-CD40L antibody or an IgG control (300 µg/week). The treatments altered the immune cell composition of adipose tissue in obese mice. Treated mice demonstrated a marked reduction in pro-inflammatory adipose tissue macrophages and activated CD8+ T cells. Mice treated with anti-CD40L exhibited reduced weight gain, which was accompanied by a trend toward improved IR. CTLA-4 Ig treatment, however, was not associated with improved IR. These data suggest that the presence of pro-inflammatory T cells and macrophages can be altered with co-stimulatory inhibitors, but may not be a significant contributor to the whole body IR phenotype.

Dendritic cells promote macrophage infiltration and comprise a substantial proportion of obesity-associated increases in CD11c+ cells in adipose tissue and liver.

Obesity-associated increases in adipose tissue (AT) CD11c(+) cells suggest that dendritic cells (DC), which are involved in the tissue recruitment and activation of macrophages, may play a role in determining AT and liver immunophenotype in obesity. This study addressed this hypothesis. With the use of flow cytometry, electron microscopy, and loss-and-gain of function approaches, the contribution of DC to the pattern of immune cell alterations and recruitment in obesity was assessed. In AT and liver there was a substantial, high-fat diet (HFD)-induced increase in DC. In AT, these increases were associated with crown-like structures, whereas in liver the increase in DC constituted an early and reversible response to diet. Notably, mice lacking DC had reduced AT and liver macrophages, whereas DC replacement in DC-null mice increased liver and AT macrophage populations. Furthermore, delivery of bone marrow-derived DC to lean wild-type mice increased AT and liver macrophage infiltration. Finally, mice lacking DC were resistant to the weight gain and metabolic abnormalities of an HFD. Together, these data demonstrate that DC are elevated in obesity, promote macrophage infiltration of AT and liver, contribute to the determination of tissue immunophenotype, and play a role in systemic metabolic responses to an HFD.