CD40-Mediated Activation of the NF-κB2 Pathway

CD40 is a critical stimulatory receptor on antigen-presenting cells of the immune system. CD40-mediated activation of B cells is particularly important for normal humoral immune function. Engagement of CD40 by its ligand, CD154, on the surface of activated T cells initiates a variety of signals in B cells including the activation of MAP kinases and NF-κB. The transcriptional regulator NF-κB is in reality a family of factors that can promote B cell activation, differentiation, and proliferation. Complex – and only partially understood – biochemical mechanisms allow CD40 to trigger two distinct NF-κB activation pathways resulting in the activation of canonical (NF-κB1) and non-canonical (NF-κB2) NF-κB. This brief review provides a summary of mechanisms responsible for activation of the latter, which appears to be particularly important for enhancing the viability of B cells at various stages in their life cycle and may also contribute to the development of B cell malignancies. CD40 is also expressed by various cell types in addition to B cells, including T cells, macrophages, dendritic cells, as well as certain non-hematopoietic cells. Here too, while perhaps less extensively studied than in B cells, the CD40-mediated activation of NF-κB2 also appears to have important roles in cellular physiology

TRAF3 as a Multifaceted Regulator of B Lymphocyte Survival and Activation

The adaptor protein TNF receptor-associated factor 3 (TRAF3) serves as a powerful negative regulator in multiple aspects of B cell biology. Early in vitro studies in transformed cell lines suggested the potential of TRAF3 to inhibit signaling by its first identified binding receptor, CD40. However, because the canonical TRAF3 binding site on many receptors also mediates binding of other TRAFs, and whole-mouse TRAF3 deficiency is neonatally lethal, an accurate understanding of TRAF3's specific functions was delayed until conditional TRAF3-deficient mice were produced. Studies of B cell-specific TRAF3-deficient mice, complemented by investigations in normal and malignant mouse and human B cells, reveal that TRAF3 has powerful regulatory roles that are unique to this TRAF, as well as functions context-specific to the B cell. This review summarizes the current state of knowledge of these roles and functions. These include inhibition of signaling by plasma membrane receptors, negative regulation of intracellular receptors, and restraint of cytoplasmic NF- κB pathways. TRAF3 is also now known to function as a resident nuclear protein, and to impact B cell metabolism. Through these and additional mechanisms TRAF3 exerts powerful restraint upon B cell survival and activation. It is thus perhaps not surprising that TRAF3 has been revealed as an important tumor suppressor in B cells. The many and varied functions of TRAF3 in B cells, and new directions to pursue in future studies, are summarized and discussed here

Requirement for TRAF3 in Signaling by LMP1 But Not CD40 in B Lymphocytes

CD40, a member of the tumor necrosis factor receptor family, and the Epstein-Barr virus–encoded oncoprotein latent membrane protein 1 (LMP1) share several tumor necrosis factor receptor–associated factor (TRAF) adaptor proteins for signaling. Among these, TRAF3 was the first identified to directly bind both receptors, yet its role remains a mystery. To address this, we generated B cell lines deficient in TRAF3 by homologous recombination. We found that CD40 signals were normal in the absence of TRAF3, with the exception of moderately enhanced c-Jun NH2-terminal kinase (JNK) activation and antibody secretion. In sharp contrast, LMP1 signaling was markedly defective in TRAF3−/− B cells. LMP1-induced activation of JNK and nuclear factor κB, up-regulation of CD23 and CD80, and antibody secretion were substantially affected by TRAF3 deficiency. Reconstitution of TRAF3 expression decreased CD40-induced JNK activation and antibody secretion, and fully restored LMP1 signaling. Although TRAF2 is widely believed to be important for LMP1 function, LMP1 signaling was intact in TRAF2−/− B cells. Our data reveal that CD40 and LMP1 unexpectedly use TRAF3 in different ways, and that TRAF3 is required for LMP1-mediated activation of B cells

Utilizing a Community-University Partnership to Meet Grandfamilies’ Needs: Development and Evaluation of a Grandchildren-mentoring Program

In response to the increasing rate of grandparent-headed homes and the needs of grandparents and grandchildren, we engaged in a community-university partnership to develop, implement, and evaluate a grandchildren-mentoring program. Prior to developing the grandchildren-mentoring program, a county needs assessment was conducted. The needs assessment revealed that grandparents wanted opportunities for mentorship of their grandchildren. As a result, we partnered with an undergraduate practicum course to create a new option of college students engaging with grandchildren. During our inaugural grandchildren-mentoring program, we conducted focus groups/interviews with grandparents (n = 5) and grandchildren (n = 7) at the end of the nine-month program. Data analysis of the focus group transcripts revealed that grandparents believed the two-hour weekly meetings (between the grandchildren and the student mentor) provided respite and grandparents would enjoy having a student again. Grandchildren commented they trusted their student mentors and did not like that they could not see them after the program ended. The grandchild-mentoring program was well received by grandparents and grandchildren and, although it was designed to support grandchildren, the program met a stated need of grandfamilies in our community. As a result, this program may be beneficial to both grandparents and grandchildren and other university-community partnerships should consider working with students to implement similar programming to support grandfamilies

Enhancement of human natural killer cells by interferon requires RNA and protein synthesis

An enriched population of human natural killer (NK) cells was obtained by density gradient centrifugation. The cytotoxic activity of these cells was enhanced by pretreatment with human leukocyte interferon (IF), and the metabolic requirements of this enhancement were examined. Augmentation of NK activity was initiated in a rapid, temperature-independent manner, requiring only a 10- to 15-min exposure to IF, and occurred at either 4 or 37[deg]C. Increased activity of the IF-treated NK effector cells was consistently observed after only 30 min of contact with target cells. Augmentation was inhibited by prior treatment of NK effector cells with actinomycin D (AD), but treatment with AD after 1 hr of IF treatment did not inhibit the IF-mediated increase in cytotoxicity, suggesting that the RNA species required for enhancement are synthesized within 1 hr of cell-IF interaction. Protein synthesis was required for at least 1 hr following cell-IF interaction, as shown by the ability of emetine and puromycin treatments to abrogate increased NK cell activity. Binding of IF to cells was independent of protein synthesis. IF-induced enhancement was unaffected by incubation of effector cells with mitomycin C either before or after IF treatment, indicating that IF acts primarily upon a population of preexisting cells of lower NK activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23787/1/0000025.pd

Tumor Necrosis Factor Receptor-Associated Factor 3 Is a Critical Regulator of B Cell Homeostasis in Secondary Lymphoid Organs

SummaryTumor necrosis factor receptor-associated factor 3 (TRAF3) is an adaptor protein that directly binds to a number of receptors of the tumor necrosis factor receptor (TNF-R) superfamily. Despite in vitro evidence that TRAF3 plays diverse roles in different cell types, little is known about the in vivo functions of TRAF3. To address this gap in knowledge and to circumvent the early lethal effect of TRAF3 null mutations, we generated conditional TRAF3-deficient mice. B-cell-specific Traf3−/− mice displayed severe peripheral B cell hyperplasia, which culminated in hyperimmunoglobulinemia and increased T-independent antibody responses, splenomegaly and lymphadenopathy. Resting splenic B cells from these mice exhibited remarkably prolonged survival ex vivo independent of B cell activating factor and showed increased amounts of active nuclear factor-κB2 but decreased amounts of nuclear protein kinase Cδ. Furthermore, these mice developed autoimmune manifestations as they aged. These findings indicate that TRAF3 is a critical regulator of peripheral B cell homeostasis and may be implicated in the regulation of peripheral self-tolerance induction

Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells

AbstractPiperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMycEμ. PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21Cip1-encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1–NF-κB–Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers

Fenfluramine treatment is associated with improvement in everyday executive function in preschool-aged children (<5 years) with Dravet syndrome: A critical period for early neurodevelopment

OBJECTIVE: To evaluate whether fenfluramine (FFA) is associated with improvement in everyday executive function (EF)-self-regulation-in preschool-aged children with Dravet syndrome (DS). METHODS: Children with DS received placebo or FFA in one of two phase III studies (first study: placebo, FFA 0.2 mg/kg/day, or FFA 0.7 mg/kg/day added to stiripentol-free standard-of-care regimens; second study: placebo or FFA 0.4 mg/kg/day added to stiripentol-inclusive regimens). Everyday EF was evaluated at baseline and Week 14-15 for children aged 2-4 years with parent ratings on the Behavior Rating Inventory of Executive Function®-Preschool (BRIEF®-P); raw scores were transformed to T-scores and summarized in Inhibitory Self-Control Index (ISCI), Flexibility Index (FI), Emergent Metacognition Index (EMI), and Global Executive Composite (GEC). Clinically meaningful improvement and worsening were defined using RCI ≥ 90% and RCI ≥ 80% certainty, respectively. The associations between placebo vs FFA combined (0.2, 0.4, and 0.7 mg/kg/day) or individual treatment groups and the likelihood of clinically meaningful change in BRIEF®-P indexes/composite T-scores were evaluated using Somers'd; pairwise comparisons were calculated by 2-sided Fisher's Exact tests (p ≤ 0.05) and Cramér's V. RESULTS: Data were analyzed for 61 evaluable children of median age 3 years (placebo, n = 22; FFA 0.2 mg/kg/day, n = 15; 0.4 mg/kg/day [with stiripentol], n = 10; 0.7 mg/kg/day, n = 14 [total FFA, n = 39]). Elevated or problematic T-scores (T ≥ 65) were reported in 55% to 86% of patients at baseline for ISCI, EMI, and GEC, and in ∼33% for FI. Seventeen of the 61 children (28%) showed reliable, clinically meaningful improvement (RCI ≥ 90% certainty) in at least one BRIEF®-P index/composite, including a majority of the children in the FFA 0.7 mg/kg/day group (9/14, 64%). Only 53% of these children (9/17) also experienced clinically meaningful reduction (≥50%) in monthly convulsive seizure frequency, including 6/14 patients in the FFA 0.7 mg/kg/day group. Overall, there were positive associations between the four individual treatment groups and the likelihood of reliable, clinically meaningful improvement in all BRIEF®-P indexes/composite (ISCI, p = 0.001; FI, p = 0.005; EMI, p = 0.040; GEC, p = 0.002). The FFA 0.7 mg/kg/day group showed a greater likelihood of reliable, clinically meaningful improvement than placebo in ISCI (50% vs 5%; p = 0.003), FI (36% vs 0%; p = 0.005), and GEC (36% vs 0%; p = 0.005). For EMI, the FFA 0.7 mg/kg/day group showed a greater likelihood of reliable, clinically meaningful improvement than the FFA 0.2 mg/kg/day group (29% vs 0%; p = 0.040), but did not meet the significance threshold compared with placebo (29% vs 5%; p = 0.064). There were no significant associations between treatment and the likelihood of reliable, clinically meaningful worsening (p > 0.05). SIGNIFICANCE: In this preschool-aged DS population with high baseline everyday EF impairment, FFA treatment for 14-15 weeks was associated with dose-dependent, clinically meaningful improvements in regulating behavior, emotion, cognition, and overall everyday EF. These clinically meaningful improvements in everyday EF were not entirely due to seizure frequency reduction, suggesting that FFA may have direct effects on everyday EF during the early formative years of neurodevelopment