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Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

Summary Macrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca2+ signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation., Graphical Abstract, Highlights • We identified a gene network that regulates macrophage multinucleation and includes Kcnn4 • Kcnn4 can be targeted in two inflammatory conditions with macrophage multinucleation • Kcnn4 regulates bone mass under physiological conditions • Kcnn4 is a drug target for which inhibitors reached phase III of clinical trials, Kang et al. establish the genetic determinants of macrophage multinucleation, which is involved in the formation of osteoclasts in bone or multinucleated giant cells during inflammatory reaction. They identify genetic variation on rat chromosome 9 linked to the expression of Kcnn4 as part of a macrophage multinucleation gene network. Kcnn4 is a calcium-activated potassium channel, and the authors show that it regulates macrophage multinucleation and bone mass. Blockade of Kcnn4 results in reduced susceptibility to glomerulonephritis and arthritis.

Objective Endothelial cells (ECs) lining arteries respond to laminar shear stress (LSS) by suppressing pro-inflammatory changes, in part through the activation of MEK5, ERK5 and induction of KLF4. We examined if this anti-inflammatory pathway operates in human ECs lining microvessels, the principal site of inflammatory responses.

PIM (proviral integration site) kinases are a distinct class of serine/threonine-specific kinases consisting of PIM1, PIM2 and PIM3. PIM2 is known to function in apoptosis pathways. Expression of PIM2 is highly induced by pro-inflammatory stimuli but the role of PIM2 in the expression of pro-inflammatory cytokines is unclear. In this study, we showed that over-expression of PIM2 in HeLa cells as well as in human umbilical vein endothelial cells enhanced interleukin-1β (IL-1β) -induced and tumour necrosis factor-α-induced IL-6 expression, whereas over-expression of a kinase-dead PIM2 mutant had the opposite effect. Studies with small interfering RNA specific to PIM2 further confirmed that IL-6 expression in HeLa cells requires PIM2. To investigate the function of PIM2 further, we generated PIM2-deficient mice. It was found that IL-6 production was significantly decreased from PIM2-deficient spleen cells after stimulation with lipopolysaccharide. Taken together, we demonstrated an important function of PIM2 in controlling the expression of the pro-inflammatory cytokine IL-6. PIM2 inhibitors may be beneficial for IL-6-mediated diseases such as rheumatoid arthritis.

CD4+ Th1 cells produce IFN-γ, TNF-α, and IL-2. These Th1 cytokines play critical roles in both protective immunity and inflammatory responses. In this study we report that sphingosine kinase 1 (SPHK1), but not SPHK2, is highly expressed in DO11.10 Th1 cells. The expression of SPHK1 in Th1 cells requires TCR signaling and new protein synthesis. SPHK1 phosphorylates sphingosine to form sphingosine-1-phosphate. Sphingosine-1-phosphate plays important roles in inhibition of apoptosis, promotion of cell proliferation, cell migration, calcium mobilization, and activation of ERK1/2. When SPHK1 expression was knocked down by SPHK1 short interfering RNA, the production of IL-2, TNF-α, and IFN-γ by Th1 cells in response to TCR stimulation was enhanced. Consistently, overexpression of dominant-negative SPHK1 increased the production of IL-2, TNF-α, and IFN-γ in Th1 cells. Furthermore, overexpression of SPHK1 in Th1 and Th0 cells decreased the expression of IL-2, TNF-α, and IFN-γ. Several chemokines, including Th2 chemokines CCL17 and CCL22, were up-regulated by SPHK1 short interfering RNA and down-regulated by overexpression of SPHK1. We also showed that Th2 cells themselves express CCL17 and CCL22. Finally, we conclude that SPHK1 negatively regulates the inflammatory responses of Th1 cells by inhibiting the production of proinflammatory cytokines and chemokines.

NF-kappaB transcription factors induce a host of genes involved in pro-inflammatory/stress-like responses; but the collateral effects and consequences of sustained NF-kappaB activation on other cellular gene expression programming remain less well understood. Here enforced expression of a constitutively active IKKbeta T-loop mutant (IKKbetaca) drove murine fibroblasts into transient growth arrest that subsided within 2-3 weeks of continuous culture. Proliferation arrest was associated with a G1/S phase block in immortalized and primary early passage MEFs. Molecular analysis in immortalized MEFs revealed that inhibition of cell proliferation in the initial 1-2 weeks after their IKKbetaca retroviral infection was linked to the transient, concerted repression of essential cell cycle effectors that are known targets of either E2F or FoxM1. Co-expression of a phosphorylation resistant IkappaBalpha super repressor and IKKbetaca abrogated growth arrest and cell cycle effector repression, thereby linking IKKbetaca's effects to canonical NF-kappaB activation. Transient growth arrest of IKKbetaca cells was associated with enhanced p21 (cyclin-dependent kinase inhibitor 1A) protein expression, due in part to transcriptional activation by NF-kappaB and also likely due to strong repression of Skp2 and Csk1, both of which are FoxM1 direct targets mediating proteasomal dependent p21 turnover. Ablation of p21 in immortalized MEFs reduced their IKKbetaca mediated growth suppression. Moreover, trichostatin A inhibition of HDACs alleviated the repression of E2F and FoxM1 targets induced by IKKbetaca, suggesting chromatin mediated gene silencing in IKKbetaca's short term repressive effects on E2F and FoxM1 target gene expression.

As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis.

In a last paper (Hu, L.; Adeyiga, A. A. 1997, 36 (6), 2375), we reported the extraction of formic acid from the reaction of sodium formate with carbon dioxide by organic tertiary amine. The process can be described by the reactions HCOONa(w)+CO 2 (g)+H 2 O(w)===NaHCO 3 (s,w)+HCOOH(w) and HCOOH(w)+NR 3 (o)===HCOOHNR 3 (o). We defined the above process as a decomposition-extraction process. The studies investigated the mechanism of the decomposition-extraction process. By engineering analysis of the process, it was found that the mass transfer of carbon dioxide from the gas phase to the aqueous phase (the aqueous solution of sodium formate or calcium chloride) proceeded essentially through the organic phase (organic tertiary amine with diluent 2-ethylhexyl alcohol) rather than directly. Then carbon dioxide in the organic phase reacted with salt in the aqueous solution to form MHCO 3 and complex NR 3 -HOOCH. The organic phase was both the extractant of formic acid and the transporter of carbon dioxide. This mechanism has been identified through the experimentation for the first time.

NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP), and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs (e. g., Crohn's disease, asthma, and atopic eczema). It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2(wt) or NOD2(L1007fsinsC) to stimulation with MDP. Importantly, a clear loss of function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, whereas the NOD2(wt) cells showed differential regulation of growth factors, chemokines, and several antagonists of NF-kappa B (e. g., TNFAIP3 A20 and IER3). This genotype-dependent regulation pattern was confirmed in primary human myelomonocytic cells. The influence of TNFAIP3 and IER3 in the context of NOD2 signaling was characterized, and we could validate the predicted role as inhibitors of NOD2-induced NF-kappa B activation. We show that IER3 impairs the protective effect of NOD2(wt) against bacterial cytoinvasion. These results further our understanding of NOD2 as a first-line defense molecule and emphasize the importance of simultaneous upregulation of counter-regulatory anti-inflammatory factors as an integral part of the NOD2-induced cellular program. Lack of these regulatory events due to the L1007fsinsC variant may pivotally contribute to the induction and perpetuation of chronic inflammation. The Journal of Immunology, 2011, 186: 4027-4038.

ECs lining arteries respond to LSS by suppressing pro-inflammatory changes, in part through the activation of MEK5, ERK5 and induction of KLF4. We examined if this anti-inflammatory pathway operates in human ECs lining microvessels, the principal site of inflammatory responses.We used immunofluorescence microscopy of human skin to assess ERK5 activation and KLF4 expression in HDMECs in situ. We applied LSS to or overexpressed MEK5/CA in cultured HDMECs and assessed gene expression by microarrays and qRT-PCR and protein expression by Western blotting. We assessed effects of MEK5/CA on TNF responses using qRT-PCR, FACS and measurements of HDMEC monolayer electrical resistance. We used siRNA knockdown to assess the role of ERK5 and KLF4 in these responses.ERK5 phosphorylation and KLF4 expression is observed in HDMECs in situ. LSS activates ERK5 and induces KLF4 in cultured HDMECs. MEK5/CA-transduced HDMECs show activated ERK5 and increased KLF4, thrombomodulin, eNOS, and ICAM-1 expression. MEK5 induction of KLF4 is mediated by ERK5. MEK5/CA-transduced HDMECs are less responsive to TNF, an effect partly mediated by KLF4.MEK5 activation by LSS inhibits inflammatory responses in microvascular ECs, in part through ERK5-dependent induction of KLF4.

PIM (proviral integration site) kinases are a distinct class of serine/threonine-specific kinases consisting of PIM1, PIM2 and PIM3. PIM2 is known to function in apoptosis pathways. Expression of PIM2 is highly induced by pro-inflammatory stimuli but the role of PIM2 in the expression of pro-inflammatory cytokines is unclear. In this study, we showed that over-expression of PIM2 in HeLa cells as well as in human umbilical vein endothelial cells enhanced interleukin-1β (IL-1β) -induced and tumour necrosis factor-α-induced IL-6 expression, whereas over-expression of a kinase-dead PIM2 mutant had the opposite effect. Studies with small interfering RNA specific to PIM2 further confirmed that IL-6 expression in HeLa cells requires PIM2. To investigate the function of PIM2 further, we generated PIM2-deficient mice. It was found that IL-6 production was significantly decreased from PIM2-deficient spleen cells after stimulation with lipopolysaccharide. Taken together, we demonstrated an important function of PIM2 in controlling the expression of the pro-inflammatory cytokine IL-6. PIM2 inhibitors may be beneficial for IL-6-mediated diseases such as rheumatoid arthritis.

Epstein-Barr virus-induced gene 3 (EBI3) associates with p28 to form IL-27 and with IL-12p35 to form IL-35. IL-27Ralpha(-/-) mice studies indicate that IL-27 negatively regulates Th17 cell differentiation. However, no EBI3, p28 or p35-deficiency studies that directly address the role of EBI3, p28 or p35 on Th17 cells have been done. Here, we demonstrate that spleen cells derived from EBI3(-/-) mice produce significantly higher levels of IL-17 as well as IL-22 upon stimulation with OVA. In vitro derived EBI3(-/-) Th17 cells also produced significantly higher levels of IL-17 and IL-22 than WT cells. The frequency of IL-17-producing cells was also elevated when EBI3(-/-) cells were cultured under Th17 conditions. In addition, spleen cells from EBI3(-/-) mice immunized with Listeria monocytogenes produced significantly elevated levels of IL-17 and IL-22. Furthermore, the Th17 transcription factor RORgamma t was significantly enhanced in EBI3(-/-) cells. Finally, EBI3(-/-) mice exhibited a reduced bacterial load following an acute challenge with L. monocytogenes or a re-challenge of previously immunized mice, suggesting that EBI3 negatively regulates both innate and adaptive immunity. Taken together, these data provide direct evidence that EBI3 negatively regulates the expression of IL-17, IL-22 and RORgamma t as well as protective immunity against L. monocytogenes.

Protein kinase C theta (PKCtheta) is essential for T cell activation, as it is required for the activation of NF-kappaB and expression of IL-2. PKCtheta has also been shown to affect NFAT activation and Th2 differentiation. To better understand the role of PKCtheta in the regulation of T helper cells, we used PKCtheta-deficient DO11.10 transgenic T cells to study its role in vitro. DO11.10 Th1 cells deficient in PKCtheta produced significantly less TNF-alpha and IL-2. The expression of Th2 cytokines, including IL-4, IL-5, IL-10, IL-13 and IL-24 was significantly reduced in PKCtheta-deficient T cells. Moreover, the expression of the Th2 transcription factor, GATA3, was significantly reduced in PKCtheta-deficient T cells. Overexpression of GATA3 by retroviral infection in PKCtheta-deficient T cells resulted in increased expansion of IL-4-producing T cells and higher IL-4 production than that of wild type Th2 cells. IL-5, IL-10, IL-13 and IL-24 expressions were also rescued by GATA3 overexpression. Our observations suggest that PKCtheta regulates Th2 cytokine expression via GATA3.

Mechatronics curricula are being established at several universities associated with the Synthesis Coalition, a National Science Foundation project. aimed at improving engineering education and increasing the number, quality and diversity of engineering graduates. The programs focus on real time computation and control of mechanical systems such as DC motors, stepping motors, thermal systems, and manufacturing machinery.

Cellular responses to stress-like stimuli require the IkappaB kinase (IKK) signalsome (IKKalpha, IKKbeta, and NEMO/IKKgamma) to activate NF-kappaB-dependent genes. IKKbeta and NEMO/IKKgamma are required to release NF-kappaB p65/p50 heterodimers from IkappaBalpha, resulting in their nuclear migration and sequence-specific DNA binding; but IKKalpha was found to be dispensable for this initial phase of canonical NF-kappaB activation. Nevertheless, IKKalpha-/- mouse embryonic fibroblasts (MEFs) fail to express NF-kappaB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKalpha in NF-kappaB activation. However, it remains unknown whether the global defect in NF-kappaB-dependent gene expression of IKKalpha-/- cells is caused by the absence of IKKalpha kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKalpha in IKKalpha-/- MEFs globally restores expression of their canonical NF-kappaB target genes. To prove that the kinase activity of IKKalpha was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKalpha mutant (IKKalpha(K44M)). Remarkably, the IKKalpha(K44M) protein rescued approximately 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-alpha responsiveness. Thus the IKKalpha-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKalpha/NF-kappaB-dependent genes.

In this paper a preliminary study of the impact of a pre-engineering program at Hampton University is presented. Data collected over a period of six years show that a large number of the electrical engineering students who go through the program are from this group. Data also shows that some of the students who went through the pre-engineering program graduated at the top of their class.

There have been considerable efforts over the past two decades to increase the retention and graduation rates and quality of women and under represented minorities in engineering. One of the approaches taken at Hampton University to achieve this goal is the Academic Enrichment Summer Program (AESP). The program is designed to give students a head-start in the field of engineering at Hampton University. The impact of the result will address the major shortfall of freshman's interest in engineering careers. The issues surrounding the significant decline and a low enrolment in engineering programs by women and under-represented minorities is discussed. The results of the cumulative five year Academic Enrichment Summer Program are presented. The participants of this program have benefited tremendously in improving their academic standing and were better prepared to start their engineering education at Hampton University when they enrolled.

A simple computer based learning tool to estimate the dielectric constant of dielectric materials is presented. This is intended to give the students both experimental and computational experience. The dielectric material is placed in a waveguide for measuring the reflection coefficient. This measured data is used as an input to the computational code utilizing the finite element method to estimate the dielectric constant. This procedure will assist students in grasping the experimental details and at the same time expose them to computational analysis.

The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKKalpha subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKKalpha's physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKKalpha is also a global regulator of tumor necrosis factor alpha- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, beta(2)-microglobulin, IL-1alpha, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKKalpha or IKKbeta. Constitutive expression of a trans-dominant IkappaBalpha superrepressor (IkappaBalphaSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (alpha and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling.

A simple waveguide measurement technique is presented to determine the complex permittivity of printed circuit board material. The printed circuit board with metal coating removed from both sides and cut into size which is the same as the cross section of the waveguide is loaded in a short X-band rectangular waveguide. Using a network analyzer, the reflection coefficient of the shorted waveguide(loaded with the sample) is measured. Using the Finite Element Method(FEM) the exact reflection coefficient of the shorted wavguide(loaded with the sample) is determined as a function of dielectric constant. Matching the measured value of the reflection coefficient with the reflection value calculated using FEM and utilizing Newton-Raphson Method, an estimate of the dielectric constant of a printed circuit board material is obtained. A comparison of estimated values of permittivity constant obtained using the present approach with the available data.