Foxo3 Promotes Apoptosis of B Cell Receptor-Stimulated Immature B Cells, Thus Limiting the Window for Receptor Editing.

Central tolerance checkpoints are critical for the elimination of autoreactive B cells and the prevention of autoimmunity. When autoreactive B cells encounter their Ag at the immature B cell stage, BCR cross-linking induces receptor editing, followed by apoptosis if edited cells remain autoreactive. Although the transcription factor Foxo1 is known to promote receptor editing, the role of the related factor Foxo3 in central B cell tolerance is poorly understood. We find that BCR-stimulated immature B cells from Foxo3-deficient mice demonstrate reduced apoptosis compared with wild type cells. Despite this, Foxo3-/- mice do not develop increased autoantibodies. This suggests that the increased survival of Foxo3-/- immature B cells allows additional rounds of receptor editing, resulting in more cells "redeeming" themselves by becoming nonautoreactive. Indeed, increased Igλ usage and increased recombining sequence recombination among Igλ-expressing cells were observed in Foxo3-/- mice, indicative of increased receptor editing. We also observed that deletion of high-affinity autoreactive cells was intact in the absence of Foxo3 in the anti-hen egg lysozyme (HEL)/membrane-bound HEL model. However, Foxo3 levels in B cells from systemic lupus erythematosus (SLE) patients were inversely correlated with disease activity and reduced in patients with elevated anti-dsDNA Abs. Although this is likely due in part to increased B cell activation in these SLE patients, it is also possible that low-affinity B cells that remain autoreactive after editing may survive inappropriately in the absence of Foxo3 and become activated to secrete autoantibodies in the context of other SLE-associated defects.

Autoreactive CD19+CD20- Plasma Cells Contribute to Disease Severity of Experimental Autoimmune Encephalomyelitis.

The contribution of autoantibody-producing plasma cells in multiple sclerosis (MS) remains unclear. Anti-CD20 B cell depletion effectively reduces disease activity in MS patients, but it has a minimal effect on circulating autoantibodies and oligoclonal bands in the cerebrospinal fluid. Recently we reported that MEDI551, an anti-CD19 mAb, therapeutically ameliorates experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. MEDI551 potently inhibits pathogenic adaptive immune responses, including depleting autoantibody-producing plasma cells. In the present study, we demonstrated that CD19 mAb treatment ameliorates EAE more effectively than does CD20 mAb. Myelin oligodendrocyte glycoprotein-specific Abs and short-lived and long-lived autoantibody-secreting cells were nearly undetectable in the CD19 mAb-treated mice, but they remained detectable in the CD20 mAb-treated mice. Interestingly, residual disease severity in the CD20 mAb-treated animals positively correlated with the frequency of treatment-resistant plasma cells in the bone marrow. Of note, treatment-resistant plasma cells contained a substantial proportion of CD19(+)CD20(-) plasma cells, which would have otherwise been targeted by CD19 mAb. These data suggested that CD19(+)CD20(-) plasma cells spared by anti-CD20 therapy likely contribute to residual EAE severity by producing autoreactive Abs. In patients with MS, we also identified a population of CD19(+)CD20(-) B cells in the cerebrospinal fluid that would be resistant to CD20 mAb treatment.

Research and therapeutics-traditional and emerging therapies in systemic lupus erythematosus.

This review summarizes traditional and emerging therapies for SLE. Evidence suggests that the heterogeneity of SLE is a crucial aspect contributing to the failure of large clinical trials for new targeted therapies. A clearer understanding of the mechanisms driving disease pathogenesis combined with recent advances in medical science are predicted to enable accelerated progress towards improved SLE diagnosis and personalized approaches to treatment.

Pathways leading to an immunological disease: systemic lupus erythematosus.

SLE is a chronic autoimmune disease caused by perturbations of the immune system. The clinical presentation is heterogeneous, largely because of the multiple genetic and environmental factors that contribute to disease initiation and progression. Over the last 60 years, there have been a number of significant leaps in our understanding of the immunological mechanisms driving disease processes. We now know that multiple leucocyte subsets, together with inflammatory cytokines, chemokines and regulatory mediators that are normally involved in host protection from invading pathogens, contribute to the inflammatory events leading to tissue destruction and organ failure. In this broad overview, we discuss the main pathways involved in SLE and highlight new findings. We describe the immunological changes that characterize this form of autoimmunity. The major leucocytes that are essential for disease progression are discussed, together with key mediators that propagate the immune response and drive the inflammatory response in SLE.

Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.

Seeking balance: Potentiation and inhibition of multiple sclerosis autoimmune responses by IL-6 and IL-10.

The cytokines IL-6 and IL-10 are produced by cells of the adaptive and innate arms of the immune system and they appear to play key roles in genetically diverse autoimmune diseases such as relapsing remitting multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Whereas previous intense investigations focused on the generation of autoantibodies and their contribution to immune-mediated pathogenesis in these diseases; more recent attention has focused on the roles of cytokines such as IL-6 and IL-10. In response to pathogens, antigen presenting cells (APC), including B cells, produce IL-6 and IL-10 in order to up-or down-regulate immune cell activation and effector responses. Evidence of elevated levels of the proinflammatory cytokine IL-6 has been routinely observed during inflammatory responses and in a number of autoimmune diseases. Our recent studies suggest that MS peripheral blood B cells secrete higher quantities of IL-6 and less IL-10 than B cells from healthy controls. Persistent production of IL-6, in turn, contributes to T cell expansion and the functional hyperactivity of APC such as MS B cells. Altered B cell activity can have a profound impact on resultant T cell effector functions. Enhanced signaling through the IL-6 receptor can effectively inhibit cytolytic activity, induce T cell resistance to IL-10-mediated immunosuppression and increase skewing of autoreactive T cells to a pathogenic Th17 phenotype. Our recent findings and studies by others support a role for the indirect attenuation of B cell responses by Glatiramer acetate (GA) therapy. Our studies suggest that GA therapy temporarily permits homeostatic regulatory mechanisms to be reinstated. Future studies of mechanisms underlying dysregulated B cell cytokine production could lead to the identification of novel targets for improved immunoregulatory therapies for autoimmune diseases.

Lipoapoptosis induced by saturated free fatty acids stimulates monocyte migration: a novel role for Pannexin1 in liver cells.

Recruitment of monocytes in the liver is a key pathogenic feature of hepatic inflammation in nonalcoholic steatohepatitis (NASH), but the mechanisms involved are poorly understood. Here, we studied migration of human monocytes in response to supernatants obtained from liver cells after inducing lipoapoptosis with saturated free fatty acids (FFA). Lipoapoptotic supernatants stimulated monocyte migration with the magnitude similar to a monocyte chemoattractant protein, CCL2 (MCP-1). Inhibition of c-Jun NH2-terminal kinase (JNK) in liver cells with SP600125 blocked migration of monocytes in a dose-dependent manner, indicating that JNK stimulates release of chemoattractants in lipoapoptosis. Notably, treatment of supernatants with Apyrase to remove ATP potently inhibited migration of THP-1 monocytes and partially blocked migration of primary human monocytes. Inhibition of the CCL2 receptor (CCR2) on THP-1 monocytes with RS102895, a specific CCR2 inhibitor, did not block migration induced by lipoapoptotic supernatants. Consistent with these findings, lipoapoptosis stimulated pathophysiological extracellular ATP (eATP) release that increased supernatant eATP concentration from 5 to ~60 nM. Importantly, inhibition of Panx1 expression in liver cells with short hairpin RNA (shRNA) decreased supernatant eATP concentration and inhibited monocyte migration, indicating that monocyte migration is mediated in part by Panx1-dependent eATP release. Moreover, JNK inhibition decreased supernatant eATP concentration and inhibited Pannexin1 activation, as determined by YoPro-1 uptake in liver cells in a dose-dependent manner. These results suggest that JNK regulates activation of Panx1 channels, and provide evidence that Pannexin1-dependent pathophysiological eATP release in lipoapoptosis is capable of stimulating migration of human monocytes, and may participate in the recruitment of monocytes in chronic liver injury induced by saturated FFA.

Labyrinthine concussion following gunshot injury: A case report.

OBJECTIVE: Labyrinthine concussion due to a postauricular gunshot wound has not been well reported. DESIGN: Retrospective chart review. STUDY SAMPLE: We describe an otherwise healthy 22-year-old male who received a gunshot wound to the left mastoid and subsequently reported hearing loss and rotational vertigo. RESULTS: Audiometric testing demonstrated significant inverted scoop shaped sensorineural hearing loss. Vestibular diagnostic testing indicated a significant uncompensated left peripheral vestibulopathy. Imaging demonstrated no structural changes to the middle ear or labyrinth, suggesting that the auditory and vestibular losses noted on diagnostic examination were likely due to labyrinthine concussion. CONCLUSIONS: Labyrinthine concussion may lead to reduced vestibular reflex pathway following gunshot wounds to the temporal bone. Clinical presentation is likely to vary significantly among cases.

IL-12 selectively programs effector pathways that are stably expressed in human CD8+ effector memory T cells in vivo.

CD8(+) cytotoxic T lymphocytes play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. IL-12 and IFN-α/ß are potent "signal 3" cytokines that are involved in both effector and memory cell development. Although the majority of effector cells are eliminated as inflammation resolves, some survive within the pool of memory cells and retain immediate effector function. In this study, we demonstrate that IL-12 instructs a unique program of effector cell differentiation that is distinct from IFN-α/ß. Moreover, effector memory (T(EM)) cells within peripheral blood display many common attributes of cells differentiated in vitro in response to IL-12, including proinflammatory cytokine secretion and lytic activity. A pattern of IL-12-induced genes was identified that demarcate T(EM) from central memory cells, and the ontologies of these genes correlated precisely with their effector functions. Further, we uncovered a unique program of gene expression that was acutely regulated by IL-12 and reflected in stable gene expression patterns within T(EM), but not T central memory cells in vivo. Thus, this study directly links a selective set of IL-12-induced genes to the programming of effector functions within the stable population of human CD8(+) T(EM) cells in vivo.

American Society for Transplantation and Cellular Therapy Guidelines for Fellowship Training in Hematopoietic Cell Transplantation and Immune Effector Cell Therapy.

Rapid advances in the field of hematopoietic cell transplantation (HCT), as well as the advent of immune effector cell therapy (IEC), have resulted in an increasing number of patients undergoing these therapies and an increasing level of expertise required to manage them. Previous guidelines for the training of HCT physicians were last published in 2012. In recognition of the expanding knowledge base and increasing skill set essential to the delivery of these treatment modalities, the American Society for Transplantation and Cellular Therapy Committee on Education has updated these guidelines to reflect nearly a decade of new knowledge in the field of HCT, as well as the evolution of IEC from an experimental modality to a widely used and mainstream therapy. The resulting document reflects the Committee on Education's recommended educational structure for programs engaged in the training, evaluation, and mentorship of HCT/IEC trainees.

Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein.

Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-α secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4(+) T-cell proliferation and IFN-γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4(+) T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells.

Dysregulated expression of CXCR4/CXCL12 in subsets of patients with systemic lupus erythematosus.

OBJECTIVE: CXCR4 is a chemokine with multiple effects on the immune system. In murine lupus models, we demonstrated that monocytes, neutrophils, and B cells overexpressed CXCR4 and that its ligand, CXCL12, was up-regulated in diseased kidneys. We undertook this study to determine whether CXCR4 expression was increased in peripheral blood leukocytes from patients with systemic lupus erythematosus (SLE) and whether CXCL12 expression was increased in kidneys from patients with SLE. METHODS: Peripheral blood leukocytes from 31 SLE patients, 8 normal controls, and 9 patients with rheumatoid arthritis were prospectively analyzed by flow cytometry for CXCR4 expression. Biopsy samples (n = 14) from patients with lupus nephritis (LN) were immunostained with anti-CXCL12 antibody. RESULTS: CD19+ B cells and CD4+ T cells from SLE patients displayed a >2-fold increase (P = 0.0001) and >3-fold increase (P < 0.0001), respectively, in median CXCR4 expression compared with that in controls (n = 7-8). Moreover, CXCR4 expression on B cells was 1.61-fold higher in patients with SLE Disease Activity Index (SLEDAI) scores >10 (n = 8) than in patients with SLEDAI scores ≤10 (n = 16) (P = 0.0008), 1.71-fold higher in patients with class IV LN (n = 5) than in patients with other classes of LN (n = 7) (P = 0.02), and 1.40-fold higher in patients with active neuropsychiatric SLE (NPSLE) (n = 6) than in patients with inactive NPSLE (n = 18) (P = 0.01). CXCL12 was significantly up-regulated in the tubules and glomeruli of kidneys in patients with LN (n = 14), with the percentage of positive cells correlating positively with the severity of LN. CONCLUSION: CXCR4 appears to be up-regulated in multiple leukocyte subsets in SLE patients. The heightened expression of CXCR4 on B cells in active NPSLE and of CXCL12 in nephritic kidneys suggests that the CXCR4/CXCL12 axis might be a potential therapeutic target for SLE patients with kidney and/or central nervous system involvement.

Early gene expression changes with rush immunotherapy.

BACKGROUND: To examine whether whole genome expression profiling could reveal changes in mRNA expression of peripheral blood mononuclear cells (PBMC) from allergic patients undergoing rush immunotherapy (RIT) that might be manifest within the first few months of treatment. METHODS: For this study, PBMC from three allergic patients undergoing RIT were assessed at four timepoints: prior to RIT, at 1 week and 7 week post-RIT, during build-up and at 4 months, after establishment of a maintenance dose. PBMC mRNA gene expression changes over time were determined by oligonucleotide microarrays using the Illumina Human-6 BeadChip Platform, which simultaneously interrogates expression profiles of > 47,000 transcripts. Differentially expressed genes were identified using well-established statistical analysis for microarrays. In addition, we analyzed peripheral blood basophil high-affinity IgE receptor (Fc epsilon RI) expression and T-regulatory cell frequency as detected by expression of CD3+CD4+CD25bright cells at each timepoint using flow cytometry. RESULTS: In comparing the initial 2 timepoints with the final 2 timepoints and analyzing for genes with ≥1.5-fold expression change (p less than or equal to 0.05, BH-FDR), we identified 507 transcripts. At a 2-fold change (p less than or equal to 0.05, BH-FDR), we found 44 transcripts. Of these, 28 were up-regulated and 16 were down-regulated genes. From these datasets, we have identified changes in immunologically relevant genes from both the innate and adaptive response with upregulation of expressed genes for molecules including IL-1ß, IL-8, CD40L, BTK and BCL6. At the 4 month timepoint, we noted a downward trend in Fc epsilon RI expression in each of the three patients and increased allergen-specific IgG4 levels. No change was seen in the frequency of peripheral T-regulatory cells expressed over the four timepoints. CONCLUSIONS: We observed significant changes in gene expression early in peripheral blood samples from allergic patients undergoing RIT. Moreover, serum levels for allergen specific IgG4 also increased over the course of treatment. These studies suggest that RIT induces rapid and dynamic alterations in both innate and adaptive immunity which can be observed in the periphery of allergic patients. These alterations could be directly related to the therapeutic shift in the allergen-specific class of immunoglobulin.

Length of Stay and Health Care Utilization Among Pediatric Autologous Hematopoietic Cell Transplantation Recipients.

Autologous hematopoietic cell transplantation (autoHCT) has become a critical component in the treatment of pediatric malignancies, allowing for high-dose chemotherapy to be given safely and with greater efficacy in a subset of children at high risk for relapse. Risk factors associated with hospital length of stay (LOS) in adults undergoing autoHCT have been studied extensively; however, there is a paucity of studies describing risk factors associated with LOS and health care cost in children undergoing autoHCT. This study sought to identify factors influencing LOS and cost in pediatric autoHCT. We assessed LOS from autologous stem cell infusion from day 0 (D0) in 100 autoHCT admissions in 73 patients with malignant disease between 2007 and 2019. We evaluated demographic, pre-transplantation, post-transplantation, and socioeconomic variables to identify potential risk factors associated with LOS and cost. AutoHCT cost data were provided by the Pediatric Health Information System database. Indications for autoHCT included neuroblastoma (35.6%), brain tumor (27.4%), and relapsed lymphoma (24.7%). The median patient age was 4.88 years (range, 0.72 to 22 years), with 71% age <12 years, and the cohort was 63% male, 77% white, and 41% Hispanic. The median LOS from D0 was 19 days (range, 13 to 100 days). On multivariable analysis, age >12 years compared with 2 to 12 years (estimate, -8.9 days; 95% confidence interval [CI], -15.1 to -2.8; P = .004) and complete remission/very good partial response disease status (estimate, -5.0 days; 95% CI, -9.6 to -0.4 days; P = .031) were associated with a significantly decreased median LOS, whereas Hispanic ethnicity (estimate, +6.8 days; 95% CI, 1.1 to 12.6 days; P = .019), >5 days of fever (estimate, +7.3 days; 95% CI, 1.4 to 13.2 days; P = .015), and pediatric intensive care unit (PICU) LOS (estimate, +14.9 days; 95% CI, 1.8 to 28.0 days; P = .025) were associated with a significant increase in median LOS. The median cost per transplantation admission was $96,850 (range, $39,833 to $587,321). Multivariable analysis showed that age >12 years (estimate, -$6,776; 95% CI, -$71,787 to -$11,402; P = .007) or <2 years (estimate, -$32,426; 95% CI, -$53,507 to -$11,345; P = .003), and complete remission/very good partial response disease status (estimate, -$20,266; 95% CI, -$40,211 to -$322; P = .046) were associated with significantly decreased median cost, whereas >5 days of fever (estimate, +$58,886; 95% CI, $30,667 to $87,105; P < .001) and PICU admission (estimate, +$102,458; 95% CI, $23,843 to $181,076; P = .011) were associated with significantly increased median cost. In summary, fever and PICU stay were found to be risk factors for increased LOS and cost. Age <12 years and Hispanic ethnicity were risk factors for increased LOS, whereas age <2 years and >12 years and female sex were associated with decreased cost. Further investigation to determine specific factors influencing LOS and cost is warranted to identify potentially modifiable risks within these patient populations.

Shared signaling networks active in B cells isolated from genetically distinct mouse models of lupus.

Though B cells play key roles in lupus pathogenesis, the molecular circuitry and its dysregulation in these cells as disease evolves remain poorly understood. To address this, a comprehensive scan of multiple signaling axes using multiplexed Western blotting was undertaken in several different murine lupus strains. PI3K/AKT/mTOR (mTOR, mammalian target of rapamycin), MEK1/Erk1/2, p38, NF-kappaB, multiple Bcl-2 family members, and cell-cycle molecules were observed to be hyperexpressed in lupus B cells in an age-dependent and lupus susceptibility gene-dose-dependent manner. Therapeutic targeting of the AKT/mTOR axis using a rapamycin (sirolimus) derivative ameliorated the serological, cellular, and pathological phenotypes associated with lupus. Surprisingly, the targeting of this axis was associated with the crippling of several other signaling axes. These studies reveal that lupus pathogenesis is contingent upon the activation of an elaborate network of signaling cascades that is shared among genetically distinct mouse models and raise hope that targeting pivotal nodes in these networks may offer therapeutic benefit.

Cutting edge: a T-bet-independent role for IFN-alpha/beta in regulating IL-2 secretion in human CD4+ central memory T cells.

IL-2 is a hallmark cytokine secreted by central memory CD4(+) T cells (T(CM)). Although naive cells rapidly secrete IL-2 in response to Ag stimulation, IL-12 inhibits IL-2 secretion in daughter cells as they differentiate into Th1 cells. In this study, we uncover a unique role for IFN-alpha in regulating IL-2 secretion by human T(CM) cells. IFN-alpha synergized with IL-12 to enhance a subset of cells that secreted high and sustained levels of IL-2. These IL-2-secreting cells displayed phenotypic and functional characteristics of T(CM) and were capable of generating IFN-gamma-secreting effectors upon secondary activation. T-bet has been implicated in negatively regulating IL-2 secretion in murine T cells; however, T-bet expression did not inhibit IFN-alpha-dependent IL-2 secretion in human T(CM) cells. Thus, our results highlight a unique role for IFN-alpha in regulating the development of IL-2-secreting human T(CM) cells.

Financial impact of post-transplant complications among children undergoing allogeneic hematopoietic cell transplantation.

Complications following allogeneic hematopoietic cell transplantation (alloHCT) continue to be a significant challenge that often result in significant morbidity/mortality and increased healthcare utilization and cost. In this study, we analyzed the impact of post-alloHCT complications on healthcare utilization and cost during first year post-transplant. We analyzed data on 240 pediatric patients. Complications analyzed included kidney injury, liver injury, lung injury, viral infections, bacterial infections, fungal infections, and acute graft-versus-host disease (GVHD). Patients were divided into three groups based on the number of complications (0-1, 2-3, and >3). Cost was estimated from charges recorded in the Pediatric Health Information System database and hospital accounting records. Patients with >3 complications had higher healthcare utilization and cost, primarily driven by inpatient hospitalization and intensive care unit admissions. Multivariable analysis of risk factors identified bacteremia ($90,166, SE = 26,636, p < 0.001), lung injury ($108,529, SE = 28,196, p < 0.001), liver injury ($90,805, SE = 28,660, p = 0.002), and grade II-IV aGVHD ($137,866, SE = 28,472, p < 0.001) as associated with significantly increased cost. Our study highlights the significant impact complications have on the overall cost of alloHCT. The identification that complications associated with high morbidity (aGVHD, pulmonary disease) are also associated with the highest financial burden emphasizes the need for future research in these areas to expand management options and improve outcomes for our patients.

Fasting is neuroprotective following traumatic brain injury.

To determine the neuroprotective effect of fasting after traumatic brain injury (TBI) and to elucidate the potential underlying mechanisms, we used a controlled cortical impact (CCI) injury model to induce either a moderate or a severe injury to adult male Sprague Dawley rats. Tissue-sparing assessments were used to determine the level of neuroprotection of fasting, hypoglycemia (insulin 10 U), or ketone (1.66 mmoles/kg/day or 0.83 mmoles/kg/day; D-beta-hydroxtbutyrate) administration. Mitochondrial isolation and respiratory studies were utilized to determine the functionality of mitochondria at the site of injury. We also investigated biomarkers of oxidative stress, such as lipid/protein oxidation, reactive oxygen species (ROS) production, and intramitochondrial calcium load, as a secondary measure of mitochondrial homeostasis. We found that fasting animals for 24 hr, but not 48 hr, after a moderate (1.5 mm), but not severe (2.0 mm), CCI resulted in a significant increase in tissue sparing. This 24-hr fast also decreased biomarkers of oxidative stress and calcium loading and increased mitochondrial oxidative phosphorylation in mitochondria isolated from the site of injury. Insulin administration, designed to mimic the hypoglycemic effect seen during fasting did not result in significant tissue sparing after moderate CCI injury and in fact induced increased mortality at some injection time points. However, the administration of ketones resulted in increased tissue sparing after moderate injury. Fasting for 24 hr confers neuroprotection, maintains cognitive function, and improves mitochondrial function after moderate (1.5 mm) TBI. The underlying mechanism appears to involve ketosis rather than hypoglycemia.

UCP-mediated free fatty acid uncoupling of isolated cortical mitochondria from fasted animals: correlations to dietary modulations.

Uncoupling proteins (UCP) translocate protons from the mitochondrial intermembrane space to the matrix, thereby "uncoupling" electron transport from the production of ATP. It has been shown that these proteins are highly expressed in animals maintained on the ketogenic diet (KD). Although the exact mechanism remains unclear, it is known that these proteins are activated within a protective antireactive oxygen species (ROS) mechanism by free fatty acids (FFA). In our current studies, mitochondrial samples were probed for the presence of UCP2, which is the most ubiquitously expressed UCP isoform. We found that both traumatic brain injury and fasting upregulated the expression of UCP2, with a synergistic upregulation in fasted injured animals. We then used mitochondria from fasted naive animals to screen a number of FFA for their activation of uncoupling as well as their ability to reduce ROS. We found that arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitoleic acid, myristic acid, and butyric acid increased mitochondrial uncoupling when added after oligomycin. These FFA, along with oleic acid, also reduced ROS in mitochondria incubated with oligomycin. In order to correlate our data to KD and fasting, both of which have been shown to be neuroprotective after neurologic insult, we determined the serum levels of FFA in KD and fasted animals using gas chromatography/mass spectroscopy. We also determined brain and cerebrospinal fluid (CSF) FFA levels from fasted animals.

Strategies for controlling item exposure in computerized adaptive testing with the partial credit model.

Exposure control research with polytomous item pools has determined that randomization procedures can be very effective for controlling test security in computerized adaptive testing (CAT). The current study investigated the performance of four procedures for controlling item exposure in a CAT under the partial credit model. In addition to a no exposure control baseline condition, the Kingsbury-Zara, modified-within-.10-logits, Sympson-Hetter, and conditional Sympson-Hetter procedures were implemented to control exposure rates. The Kingsbury-Zara and the modified-within-.10-logits procedures were implemented with 3 and 6 item candidate conditions. The results show that the Kingsbury-Zara and modified-within-.10-logits procedures with 6 item candidates performed as well as the conditional Sympson-Hetter in terms of exposure rates, overlap rates, and pool utilization. These two procedures are strongly recommended for use with partial credit CATs due to their simplicity and strength of their results.