Vitamin D and Omega 3 Field Study on Progression of Type 1 Diabetes.

Chronic inflammation has been linked to the progression of type 1 diabetes (T1D). Supplementation with vitamin D and omega-3 fatty acids, which have anti-inflammatory properties, may slow or stop the progression of T1D. A field study is underway to assess the relationship between these nutrients and T1D progression among auto-antibody positive individuals who have not been diagnosed with T1D. The T1D Prevention Field Study is currently recruiting participants to complete online health surveys and home blood-spot tests for 25-hydroxyvitamin D [25(OH)D], Omega-3 Index, AA:EPA Ratio, high-sensitivity C-reactive protein, and HbA1c every three to four months for 5 years. Participants (or their parents/guardians) are given information about the importance of achieving a 25(OH)D level between 40-60 ng/ml and an AA:EPA Ratio between 1.5-3.0 to reduce inflammation. However, participants are free to choose their own supplement or dietary regimens. Data analysis will focus on associations between vitamin D and omega-3 status and progression of T1D. Initial enrollment in the T1D Prevention Field Study includes 103 participants from fifteen countries; total enrollment is expected to reach at least 400 participants by the end of 2022. The field study approach allows for cost-effective research that capitalizes on new technologies for recruitment, data collection, and blood level testing from home. However, some challenges have arisen. Many individuals are reading the open source protocols and some choose to supplement and test on their own so incentives may be needed to increase enrollment. Additionally, some participants do not have access to auto-antibody testing or are unable to get access to their test results; therefore, there is a need to provide blood spot auto-antibody testing through the field study.

Enrichment of IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets is associated with reduction of NKp44+ ILC3s in the terminal ileum of Crohn's disease patients.

Crohn's disease (CD) is a chronic inflammatory condition of the human gastrointestinal tract whose aetiology remains largely unknown. Dysregulated adaptive immune responses and defective innate immunity both contribute to this process. In this study, we demonstrated that the interleukin (IL)-17A+ interferon (IFN)-γ+ and IL-22+ IFN-γ+ T cell subsets accumulated specifically in the inflamed terminal ileum of CD patients. These cells had higher expression of Ki-67 and were active cytokine producers. In addition, their proportions within both the IL-17A-producer and IL-22-producer populations were increased significantly. These data suggest that IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets might represent the pathogenic T helper type 17 (Th17) population in the context of intestinal inflammation for CD patients. In the innate immunity compartment we detected a dramatic alteration of both phenotype and function of the intestinal innate lymphoid cells (ILCs), that play an important role in the maintenance of mucosal homeostasis. In the inflamed gut the frequency of the NKp44- CD117- ILC1s subset was increased significantly, while the frequency of NKp44+ ILC3s was reduced. Furthermore, the frequency of human leucocyte antigen D-related (HLA-DR)-expressing-NKp44+ ILC3s was also reduced significantly. Interestingly, the decrease in the NKp44+ ILC3s population was associated with an increase of pathogenic IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets in the adaptive compartment. This might suggest a potential link between NKp44+ ILC3s and the IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets in the terminal ileum of CD patients.

Micro and Nano Material Carriers for Immunomodulation.

Modulation of the immune system through the use of micro and nano carriers offers opportunities in transplant tolerance, autoimmunity, infectious disease, and cancer. In particular, polymeric, lipid, and inorganic materials have been used as carriers of proteins, nucleic acids, and small drug molecules to direct the immune system toward either suppressive or stimulatory states. Current technologies have focused on the use of particulates or scaffolds, the modulation of materials properties, and the delivery of biologics or small drug molecules to achieve a desired response. Discussed are relevant immunology concepts, the types of biomaterial carriers used for immunomodulation highlighting their benefits and drawbacks, the material properties influencing immune responses, and recent examples in the field of transplant tolerance.

Implementing personalized medicine: development of a cost-effective customized pharmacogenetics genotyping array.

Although there is increasing evidence to support the implementation of pharmacogenetics in certain clinical scenarios, the adoption of this approach has been limited. The advent of preemptive and inexpensive testing of critical pharmacogenetic variants may overcome barriers to adoption. We describe the design of a customized array built for the personalized-medicine programs of the University of Florida and Stanford University. We selected key variants for the array using the clinical annotations of the Pharmacogenomics Knowledgebase (PharmGKB), and we included variants in drug metabolism and transporter genes along with other pharmacogenetically important variants.

Pre-transplant myeloid dendritic cell deficiency associated with cytomegalovirus infection and death after kidney transplantation.

BACKGROUND: Dendritic cells (DCs) are potent antigen-presenting cells critical for immunity. We previously demonstrated a significant association between pre-transplant blood myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) deficiency and post-transplant BK viremia in renal transplant recipients. In the current post-hoc analysis, we studied the association of these same pre-transplant DC levels with other post-transplant outcomes. METHODS: Pre-transplant peripheral blood mDC and pDC levels were quantified using flow cytometry in 78 patients undergoing kidney transplantation. Post-transplant outcomes were analyzed, including infection, rejection, and patient death, with a median follow-up of 5.3 years. Associations between DC levels and outcomes were assessed using logistic regression analysis and Cox proportional hazards models. RESULTS: An independent association of mDC levels with post-transplant cytomegalovirus infection (adjusted odds ratio 7.0, P = 0.01) and patient death (adjusted hazard ratio 13.0, P = 0.015) was found. No associations were demonstrated between levels of either DC subtype and bacterial infections or rejection. CONCLUSIONS: Pre-transplant mDC deficiency is significantly associated with CMV infection and death after kidney transplantation.

Hyper-responsive phenotype in localized aggressive periodontitis.

The 'hyper-responsive' trait is an increased inflammatory response upon stimulation of innate immune receptors. Our objective was to determine if a hyper-reactive trait is present in a cohort diagnosed with aggressive periodontitis (LAgP). Peripheral blood was collected from 30 LAgP, 10 healthy unrelated, and 10 healthy sibling participants and stimulated with lipopolysaccharide (LPS) from E. coli and P. gingivalis. Cyto/chemokine response profiles were evaluated and analyzed by ANOVA. Elevated levels of pro-inflammatory cyto/chemokines were detected in E. coli and P. gingivalis LPS-stimulated LAgP cultures when compared with those of healthy unrelated control individuals. Periodontally healthy siblings presented with attenuated hyper-inflammatory cyto/chemokine profiles. Regression analysis demonstrated the hyper-reactive trait to be concomitant expression of pro-inflammatory cyto/chemokines and an absence of anti-inflammatory mediator expression. Our findings demonstrate hyper-responsive trait in a LAgP cohort, along with an attenuated hyper-responsiveness in healthy siblings, which can be induced in response to multiple TLR ligations.

Comparison between omega-3 and omega-6 polyunsaturated fatty acid intakes as assessed by a food frequency questionnaire and erythrocyte membrane fatty acid composition in young children.

OBJECTIVE: We conducted a dietary validation study in youth aged 1-11 years by comparing dietary intake of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) as assessed by a parent-completed semiquantitative food frequency questionnaire (FFQ) over time to erythrocyte membrane composition of the same fatty acids. DESIGN: The study population included youth aged 1-11 years who were participants in the Diabetes Autoimmunity Study in the Young (DAISY), a longitudinal study in Denver, Colorado that is following a cohort of youth at risk for developing type I diabetes. Four hundred and four children who had erythrocyte membrane fatty acid data matched to an FFQ corresponding to the same time frame for a total of 917 visits (matches) were included. PUFA intake was expressed both as g/day (adjusted for total energy) and as percent of total fat intake. We used mixed models to test the association and calculate the correlation between the erythrocyte membrane estimates and PUFA intake using all records of data for each youth. RESULTS: Intakes of total omega-3 fatty acids (beta=0.52, P<0.0001, rho=0.23) and marine PUFAs (beta=1.62, P<0.0001, rho=0.42), as a percent of total fat in the diet, were associated with percent of omega-3 and marine PUFAs in the erythrocyte membrane. Intakes of omega-6 PUFAs (beta=0.04, P=0.418, rho=0.05) and arachidonic acid (beta=0.31, P=0.774, rho=0.01) were not associated. CONCLUSIONS: In these young children, an FFQ using parental report provided estimates of average long-term intakes of marine PUFAs that correlated well with their erythrocyte cell membrane fatty acid status.

Dexamethasone induces IL-10-producing monocyte-derived dendritic cells with durable immaturity.

It is highly desirable that immature dendritic cells (DC) used for tolerance induction maintain steady immature state with predominant interleukin (IL)-10 production. In this study, we attempted to develop DC with durable immaturity and other tolerogenic features by using dexamethasone (Dex). We found DC derived from human monocytes in the presence of 10(-7) m Dex were negative for CD1a. Compared with control transduced DC (Ctrl-DC), Dex-DC expressed lower CD40, CD80 and CD86 but equivalent human leucocyte antigen-DR. Both immature Dex- and Ctrl-DC did not express CD83. Nevertheless, upon stimulation of lipopolysaccharide (LPS) or CD40 ligand, the expression of CD40, CD80, CD83 and CD86 was upregulated on Ctrl-DC but not on Dex-DC. The immaturity of Dex-DC was durable following Dex removal. Interestingly, Dex-DC maintained production of large amount of IL-10 and little IL-12 five days after Dex removed. Further study indicated that high-level IL-10 production by Dex-DC was associated with high-level phosphorylation of extracellular signal-regulated kinase (ERK) as blockade of this enzyme markedly attenuated IL-10 production. Furthermore, Dex-DC sustained the capability of high phosphorylation of ERK and IL-10 production 5 days after Dex removal. In addition, Dex-DC had significantly lower activity in stimulating T-cell proliferation. Neutralization of IL-10, to some extent, promoted DC maturation activated by LPS, as well as T-cell stimulatory activity of Dex-DC. The above findings suggest that IL-10-producing Dex-DC with durable immaturity are potentially useful for induction of immune tolerance.

Signal transduction activator of transcription 5 (STAT5) dysfunction in autoimmune monocytes and macrophages.

Autocrine granulocyte macrophage-colony stimulating factor (GM-CSF) sequentially activates intracellular components in monocyte/macrophage production of the pro-inflammatory and immunoregulatory prostanoid, prostaglandin E2 (PGE2). GM-CSF first induces STAT5 signaling protein phosphorylation, then prostaglandin synthase 2 (COX2/PGS2) gene expression, and finally IL-10 production, to downregulate the cascade. Without activation, monocytes of at-risk, type 1 diabetic (T1D), and autoimmune thyroid disease (AITD) humans, and macrophages of nonobese diabetic (NOD) mice have aberrantly high GM-CSF, PGS2, and PGE2 expression, but normal levels of IL-10. After GM-CSF stimulation, repressor STAT5A and B isoforms (80-77kDa) in autoimmune human and NOD monocytes and activator STAT5A (96-94kDa) and B (94-92kDa) isoforms in NOD macrophages stay persistently tyrosine phosphorylated. This STAT5 phosphorylation persisted despite treatment in vitro with IL-10, anti-GM-CSF antibody, or the JAK2/3 inhibitor, AG490. Phosphorylated STAT5 repressor isoforms in autoimmune monocytes had diminished DNA binding capacity on GAS sequences found in the PGS2 gene enhancer. In contrast, STAT5 activator isoforms in NOD macrophages retained their DNA binding capacity on these sites much longer than in healthy control strain macrophages. These findings suggest that STAT5 dysfunction may contribute to dysregulation of GM-CSF signaling and gene activation, including PGS2, in autoimmune monocytes and macrophages.

The effects of renal transplantation on circulating precursor dendritic cells.

BACKGROUND: Dendritic cells (DCs) are potent antigen-presenting cells that induce and regulate immune responses. Recent advances allow accurate quantification of peripheral blood (PB) myeloid and plasmacytoid DC populations (mDC and pDC, respectively), although the response to renal transplantation (RT) remains unknown. METHODS: Using flow cytometry, PBDC levels were quantified in patients with end-stage renal disease (ESRD) undergoing renal transplantation. RESULTS: PBDC levels were significantly reduced in ESRD patients pretransplantation compared to healthy controls, with further reduction noted immediately following a hemodialysis session. RT resulted in a dramatic decrease in both subsets, with a greater reduction of pDC levels. Both subset levels were significantly lower than in control patients undergoing abdominal surgery without RT. Subgroup analysis revealed significantly greater mDC reduction in RT recipients receiving antilymphocyte therapy, with preferential binding of antibody preparation to this subset. Samples from later time points revealed a gradual return of PBDC levels back to pretransplant values concurrent with overall reduction of immunosuppression. Finally, PBDC levels were significantly reduced in patients with BK virus nephropathy compared to recipients with stable graft function, despite lower overall immunosuppression. CONCLUSIONS: Our findings suggest that PBDC levels may reflect the degree of immunosuppression in renal allograft recipients. Furthermore, PBDC monitoring may represent a novel strategy to predict important outcomes such as acute rejection, long-term graft loss, and infectious complications.

In vivo transduction of thymic dendritic cells with adenovirus and its potential use in acute inflammatory diseases.

Dendritic cells (DC) represent a potential target for gene therapy. In their ability to process antigens and present them to T cells, DC have been allocated a unique role as initiators of the immune response in both the innate and acquired immunity. Recent in vitro studies have showed the feasibility of DC transduction with adenoviral recombinants. In cancer therapy, targeting of DC with adenovirus has been proved to be effective in inhibiting tumour growth, as well as in reducing the number of tumour metastases. The aim of our study is to evaluate the feasibility of in vivo transduction of DC in a murine lymphocyte-rich compartment (thymus) as a potential treatment for acute inflammatory diseases. Nearly 50% of the total thymic DC were transduced with a first-generation adenoviral construct following intrathymic injection, and post-transductional inflammation was neglectable. Transduction of thymic cells with adenoviral recombinants was able to induce the expression of an intracellular protein (beta-galactosidase, green fluorescent protein), as well as the secretion of human interleukin-10, within the local compartment. Furthermore, this induction of the latter significantly decreased thymic apoptosis in the applied model of acute bacterial peritonitis (cecal ligation and puncture).

IL10 resistant PGS2 expression in at-risk/Type 1 diabetic human monocytes.

Aberrant prostaglandin synthase 2 (PGS2/COX2) expression constitutes an antigen presenting cell (APC) dysfunction seen in monocytes of humans at risk for or with Type 1 diabetes. During endotoxin activation of PGS2 expression in healthy monocytes, granulocyte-monocyte colony stimulating factor (GM-CSF) is activated and, in turn, promotes PGS2 gene activation. GM-CSF is considered a major target the action for IL10 in its suppression of PGS2. We found that the PGS2 expression in monocytes from 47% of at-risk and diabetic humans tested were highly resistant to suppression by IL10 (maintaining > or =50% of their untreated expression), and had significantly increased GM-CSF production in vitro (1043+/-SD2798 pg/10(6)cells, subject n=35, vs 29.7+/-SD91 pg/10(6)cells, control n=20; P=0.0165). The PGS2 insensitivity to IL10 of these cells was not due to a lack of IL10 functionality or its suppression of GM-CSF. In contrast to its effects on PGS2, IL10 regulation of GM-CSF and other monocyte factors (i.e., DR, IL1beta, TNFalpha, IL12, CD54, and CD64) remained intact. These findings suggest that the inability of IL10 to properly downregulate PGS2 gene expression may contribute to its dysregulation in Type 1 diabetes.

Abnormal peripheral blood dendritic cell populations in type 1 diabetes.

Type 1 diabetes (T1D) is a T cell-mediated disease. Various DC populations play important roles in initiating and directing T cell responses and thus may be critical for T1D pathogenesis. We thus examined peripheral blood DC1 and DC2 populations by flow cytometry in healthy controls, subjects at risk for T1D, new-onset patients, and established T1D patients. We found a significant increase in the number of DCs (including DC1 and DC2) in at-risk subjects and those with new-onset T1D versus healthy controls and established T1D patients (ANOVA; p < 0.0001). Analysis of DC1 and DC2 subsets in these same groups demonstrated a significant decrease in the ratio of DC1 and DC2 in subjects at risk and new-onset and established T1D patients in contrast with healthy controls (p < 0.0001). Both subsets of peripheral blood DCs from T1D patients expressed significantly higher levels of HLA-DR than healthy controls. Peripheral blood mononuclear cells from T1D patients secreted significantly higher amounts of IFN-alpha than controls, and IFN-alpha production correlated inversely with the DC1/DC2 ratio. This study demonstrates a marked increase in peripheral blood DC numbers that occurs during a time of active autoimmunity in at-risk subjects and patients with new-onset T1D, but is lost in established diabetes. However, the abnormal distribution of peripheral blood DC populations appears to be a persistent phenotype in all stages of T1D.

Defective maturation of myeloid dendritic cell (DC) in NOD mice is controlled by IDD10/17/18.

We previously demonstrated that adoptive transfer of NOD pancreatic lymph node (PLN) DC protected recipients from diabetes. Our recent studies showed that the tolerogenic DC population presented islet antigens and were mature myeloid DC that did not produce IL-12, suggestive of exhausted or fully mature DC. Extensive characterization of the DC population in vivo in NOD and control mice demonstrated a specific deficiency of PLN tolerogenic DC in older mice. These findings suggest autoimmunity might arise in NOD mice secondary to deficient maturation of myeloid DC to a tolerogenic state. To address this issue, we characterized maturation and function at development of bone marrow-derived myeloid DC from NOD and several control strains. We found that NOD DC were highly resistant to several maturation stimuli and maintained an immature phenotype (average % immature DC: 75% in NOD versus 15% in B6, p < 0.01). A survey of congenic NOD mice with various NOD diabetes susceptibility loci demonstrated that the IDD10/17/18 region on chromosome 3 controlled approximately 50% of the NOD DC maturation defect. The defect also affected NOD DC that underwent phenotypic maturation. These cells appeared to arrest in a "maturing" phase as they produced 5- to 7-fold more IL-12 than control strains and significantly less IL-10. The cytokine defect was completely corrected in NOD IDD10/17/18 mice. In addition, the IDD10/17/18 locus limited DC accumulation in islets and significantly increased tolerogenic DC in the PLN. Together, the above findings suggest that polygenic regulation of DC maturation defects in NOD mice promotes islet inflammation while limiting the generation of tolerogenic DC.

Aberrant monocyte prostaglandin synthase 2 (PGS2) expression in type 1 diabetes before and after disease onset.

METHODS: We examined monocyte prostaglandin synthase 2 (PGS2/COX2) expression in individuals at risk for or with type 1 diabetes including: (i) 58 established type 1 and 2 diabetic patients; (ii) 34 autoantibody positive (AA+) children and adults; (iii) 164 infants and young children with insulin-dependent diabetes mellitus (IDDM) susceptibility human leukocyte antigen (HLA) alleles; and (iv) 37 healthy control individuals, over a 5-yr period. RESULTS: Established type 1 diabetic patients (1 month to 30+ yr post-disease onset) had significantly higher PGS2 expression than healthy controls; by contrast, insulin-treated type 2 diabetic patients had significantly lower PGS2 expression than healthy controls. Longitudinal studies of AA+ subjects at risk for type 1 diabetes indicated that 73% (11/15) of individuals who developed this disease during the study period expressed high levels of PGS2 prior to or after onset. We also found high level PGS2 expression in genetically at-risk infants and young children that correlated with having a first-degree relative with type 1 diabetes, but not with age, gender, or HLA genotype. In this population, high level PGS2 expression coincided with or preceded autoantibody detection in 30% (3/10) of subjects. CONCLUSIONS: These findings suggest that high level monocyte PGS2 expression, although subject to fluctuation, is present in at-risk subjects at an early age and is maintained during progression to and after type 1 diabetes onset.

Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis.

Sepsis remains a significant clinical conundrum, and recent clinical trials with anticytokine therapies have produced disappointing results. Animal studies have suggested that increased lymphocyte apoptosis may contribute to sepsis-induced mortality. We report here that inhibition of thymocyte apoptosis by targeted adenovirus-induced thymic expression of human IL-10 reduced blood bacteremia and prevented mortality in sepsis. In contrast, systemic administration of an adenovirus expressing IL-10 was without any protective effect. Improvements in survival were associated with increases in Bcl-2 expression and reductions in caspase-3 activity and thymocyte apoptosis. These studies demonstrate that thymic apoptosis plays a critical role in the pathogenesis of sepsis and identifies a gene therapy approach for its therapeutic intervention.

Apoptosis in sepsis: a new target for therapeutic exploration.

The treatment of sepsis and septic shock remains a clinical conundrum, and recent prospective trials with biological response modifiers aimed at the inflammatory response have shown only modest clinical benefit. Recently, interest has shifted toward therapies aimed at reversing the accompanying periods of immune suppression. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis of lymphoid organs and some parenchymal tissues contributes to this immune suppression, anergy, and organ system dysfunction. During sepsis syndromes, lymphocyte apoptosis can be triggered by the absence of IL-2 or by the release of glucocorticoids, granzymes, or the so-called 'death' cytokines: tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via auto-activation of cytosolic and/or mitochondrial caspases, which can be influenced by the pro- and anti-apoptotic members of the Bcl-2 family. In experimental animals, not only can treatment with inhibitors of apoptosis prevent lymphoid cell apoptosis; it may also improve outcome. Although clinical trials with anti-apoptotic agents remain distant due in large part to technical difficulties associated with their administration and tissue targeting, inhibition of lymphocyte apoptosis represents an attractive therapeutic target for the septic patient.

Reduced susceptibility of nonobese diabetic mice to TNF-alpha and D-galactosamine-mediated hepatocellular apoptosis and lethality.

Nonobese diabetic (NOD/LtJ or NOD) mice are resistant to doses of LPS and D-galactosamine that uniformly produce lethality in C57BL/6J (B6) mice (p < 0.01). Liver caspase-3-like activity, serum transaminase levels (both p < 0.05), and the numbers of apoptotic liver nuclei were also reduced in NOD compared with B6 mice treated with LPS (100 ng) and D-galactosamine (8 mg). NOD mice were also at least 100-fold more resistant to recombinant human TNF-alpha and D-galactosamine treatment than B6 mice (p < 0.001). Binding of recombinant human TNF-alpha to splenocytes from NOD mice was similar to that seen in B6 mice, suggesting that the defect in responsiveness was not due to an inability of recombinant human TNF-alpha to bind the NOD TNF type 1 (p55) receptor. Because the TNF type 1 (p55) receptor shares a common signaling pathway with Fas (CD95), NOD and B6 mice were treated with the Fas agonist antibody, Jo-2. Surprisingly, NOD mice were as sensitive as B6 mice to Fas-induced lethality and hepatic injury. In addition, primary hepatocytes isolated from NOD mice and cultured in vitro in the presence of D-galactosamine with or without TNF-alpha were found to be resistant to apoptosis and cytotoxicity when compared with B6 mice. In contrast, Jo-2 treatment produced similar increases in caspase-3 activity and cytotoxicity in primary hepatocytes from NOD and B6 mice. The resistance to LPS- and TNF-alpha-mediated lethality and hepatic injury in D-galactosamine-sensitized NOD mice is apparently due to a post-TNFR binding defect, and independent of signaling pathways shared with Fas.

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production.

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.

Glucocorticoid-induced, caspase-dependent organ apoptosis early after burn injury.

Immune suppression and increased apoptotic loss of circulating lymphocytes have been reported after burn injury. However, little is known about the underlying mechanisms responsible for the increased apoptosis of lymphoid and parenchymal cells in solid organs and the role played by inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and Fas ligand (FasL), as well as by glucocorticoids. To evaluate the role of endogenously produced glucocorticoids and FasL, mice subjected to a 20% steam burn were pretreated with a glucocorticoid receptor antagonist (mifepristone) or a neutralizing murine Fas fusion protein. Three and twenty-four hours after burn injury, histological analysis, caspase-3 activity, and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and phenotyping of lymphocyte populations for apoptosis were evaluated. Burn injury increased the number of apoptotic cells and caspase-3 activity in thymus and spleen, but not in other solid organs. Increased apoptosis was seen in several T and B cell populations from both thymus and spleen. Mifepristone pretreatment significantly reduced the apoptosis and caspase-3 activity after burn injury, whereas blocking FasL activity had only minimal effects. We conclude that corticosteroids, and not FasL, are primarily responsible for the increased caspase-3 activity and apoptosis in thymus and spleen cell populations early after burn injury.