Impact of IL-1ß on lung pathology caused by Mycobacterium abscessus infection and its association with IL-17 production.

Mycobacterium abscessus (MAB), a non-tuberculous mycobacterium (NTM), causes chronic pulmonary inflammation in humans. The NLRP3 inflammasome is a multi-protein complex that triggers IL-1ß maturation and pyroptosis through the cleavage of caspase-1. In this study, we investigated the roles of NLRP3 and IL-1ß in the host's defense against MAB. The IL-1ß production by MAB was completely abolished in NLRP3, but not NLRC4, deficient macrophages. The NLRP3 inflammasome components, which are ASC and caspase-1 were also found to be essential for IL-1ß production in response to MAB. NLRP3 and IL-1ß deficiency did not affect the intracellular growth of MAB in macrophages, and the bacterial burden in lungs of NLRP3- and IL-1ß-deficient mice was also comparable to the burden observed in WT mice. In contrast, IL-1ß deficiency ameliorated lung pathology in MAB-infected mice. Notably, the lung homogenates of IL-1ß-deficient mice had reduced levels of IL-17, but not IFN-γ and IL-4 when compared with WT counterparts. Furthermore, in vitro co-culture analysis showed that IL-1ß signaling was essential for IL-17 production in response to MAB. Finally, we observed that the anti-IL-17 antibody administration moderately mitigated MAB-induced lung pathology. These findings indicated that IL-1ß production contribute to MAB-induced lung pathology via the elevation of IL-17 production.

Medicarpin Increases Antioxidant Genes by Inducing NRF2 Transcriptional Level in HeLa Cells.

The nuclear factor erythroid-derived 2-related factor 2 (NRF2) plays a pivotal role in the regulation of genes involved in oxidative stress and drug detoxification. Therefore, it is important to find NRF2 inducers to protect cells from excessive oxidative damage. Here, we investigated the effect of medicarpin isolated from the root of Robinia pseudoacacia L. on the activity of NRF2 in HeLa cells. Medicarpin significantly induced the antioxidant response elements (ARE)-luciferase activity in a concentration-dependent manner. Furthermore, medicarpin not only induced HO-1, GCLC, and NQO1 mRNA by translocating NRF2 to the nucleus but also induced the mRNA level of NRF2. To verify the NRF2 induction mechanism by medicarpin, ~2 kb of NRF2 promoter-luciferase assay was executed. As a result, medicarpin significantly induced NRF2-luciferase activity. Moreover, medicarpin strongly inhibited the ubiquitin-dependent proteasomal degradation of NRF2. Thus, medicarpin might protect cells by promoting the NRF2 transcriptional activity.

Phenotypic Differences of CD103+ Tissue-Resident Memory T Cells Associated with Various Cancers.

BACKGROUND/AIMS: The presence and clinical importance of tissue-resident memory T (TRM) cells have been recently described in association with various cancer types. However, the frequency and the traditional naïve-effector-memory phenotypic characteristics of TRM cells are largely unknown. METHODS: We analyzed single-cell populations of colorectal cancer (CC, n = 18), stomach cancer (SC, n = 13), renal cell carcinoma (RCC, n = 19), and breast cancer (BC, n = 16) by dissociation of tumor tissue with collagenase/hyaluronidase. We investigated populations of naïve, effector, and memory T and TRM cells by flow cytometry. RESULTS: Among CD8- cells, CC was associated with a significantly higher proportion of CD103+ T cells than other tumor types (p < 0.001). Among CD8+ cells, CC and SC were associated with higher CD103+ T-cell proportions than RCC and BC (p < 0.001). Significantly more CD8+ than CD8- cells expressed CD103 (p < 0.001). In association with SC, RCC, and BC, CD8+ T cells had a similar T-cell phenotype composition pattern: fewer effector T cells and more memory-type T cells among CD103+ cells compared with CD103- cells (p < 0.05). Tumors with higher proportion of CD103+ cells had no specific clinicopathologic characteristics than those with lower proportion of CD103+ cells. CONCLUSION: TRM cell abundance and phenotypes varied among CC, SC, RCC, and BC. Further studies regarding the functional differences of TRM associated with various tumors are warranted.

4-Methoxycinnamic acid attenuates schizophrenia-like behaviors induced by MK-801 in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Scrophularia buergeriana has been used for traditional medicine as an agent for reducing heat in the blood and for nourishing kidney 'Yin'. Therefore, S. buergeriana might be a potential treatment for mental illness, especially schizophrenia, which may be attenuated by supplying kidney Yin and reducing blood heat. In a pilot study, we found that S. buergeriana alleviated sensorimotor gating dysfunction induced by MK-801. AIM OF THE STUDY: In the present study, we attempted to reveal the active component(s) of S. buergeriana as a candidate for treating sensorimotor gating dysfunction, and we identified 4-methoxycinnamic acid. We explored whether 4-methoxycinnamic acid could affect schizophrenia-like behaviors induced by hypofunction of the glutamatergic neurotransmitter system. MATERIALS AND METHODS: Mice were treated with 4-methoxycinnamic acid (3, 10, or 30 mg/kg, i.g.) under MK-801-induced schizophrenia-like conditions. The effect of 4-methoxycinnamic acid on schizophrenia-like behaviors were explored using several behavioral tasks. We also used Western blotting to investigate which signaling pathway(s) is involved in the pharmacological activities of 4-methoxycinnamic acid. RESULTS: 4-Methoxycinnamic acid ameliorated MK-801-induced prepulse inhibition deficits, social interaction disorders and cognitive impairment by regulating the phosphorylation levels of PI3K, Akt and GSK-3ß signaling in the prefrontal cortex. And there were no adverse effects in terms of catalepsy and motor coordination impairments. CONCLUSION: Collectively, 4-methoxycinnamic acid would be a potential candidate for treating schizophrenia with fewer adverse effects, especially the negative symptoms and cognitive dysfunctions.

A DNA Topoisomerase II Inhibitor Results in Ex Vivo Differentiation of THP-1 Cells and Activation of Dendritic Cells.

BACKGROUND/AIM: Effective ex vivo maturation of dendritic cells (DCs) can increase the efficiency of cancer immunotherapy. We aimed to identify novel chemicals with the potential to differentiate and activate immature DCs (iDCs) to mature DCs (mDCs). MATERIALS AND METHODS: The expression of surface markers on THP-1 monocytes treated with the screened compounds was analyzed using FACS. Subsequent DC subset analysis and secreted cytokine profiling were also performed. RESULTS: FACS analysis showed that THP-1 cells treated with amsacrine hydrochloride, a DNA topoisomerase II inhibitor, exhibited the typical phenotype of conventional DCs (cDCs). The expression of DC activation markers was also increased after amsacrine treatment. The profile of cytokines produced by THP-1 cells treated with amsacrine was similar to that of mDCs. CONCLUSION: Amsacrine has an ex vivo capability of differentiating THP-1 monocytes into cDCs. As amsacrine has been used as a stable chemotherapeutic agent in humans, it can be useful for producing mDCs for cancer immunotherapy.

Surgical hip dislocation and varus derotation osteotomy for extra-articular cause of femoroacetabular impingement: a single case report.

Femoral retroversion is an extra-articular cause of cam-type femoroacetabular impingement (FAI) via early engagement with anterior rim. Valgus hip also causes extra-articular FAI by decreasing the range of motion. We present a case of valgus hip accompanied by femoral retroversion, which was refractory to prior arthroscopic femoroplasty. As a reasonable strategy, we have performed extra-articular correction via femoral subtrochanteric varus derotation osteotomy as well as intra-articular decompression by surgical hip dislocation. Femoral varus derotation osteotomy with surgical hip dislocation is a rational and appropriate solution in patients with extra-articular FAI, which is refractory to arthroscopic FAI surgery. Extra-articular causes of FAI should be suspected in every refractory case.

Clinical and Computed Tomography Angiographic Predictors of Coronary Lesions That Later Progressed to Chronic Total Occlusion.

OBJECTIVES: This study aimed to investigate clinical and coronary computed tomographic angiography (CTA) characteristics of lesions that progressed to chronic total occlusion (CTO). BACKGROUND: CTO is one of the most common reasons for referral to coronary artery bypass surgery. Prediction and adequate early management for future CTO lesions may be beneficial. METHODS: The study evaluated patients with at least 1 vessel with a diameter stenosis of ≥70% on invasive coronary angiography (ICA) who underwent previous coronary CTA >12 months before ICA, from 2006 to 2015. The study compared the baseline clinical and coronary CTA characteristics of the patients with future CTO lesions with those of the patients with future non-CTO lesions (patient-level analysis) and compared coronary CTA findings between the future CTO lesion with the most stenotic non-CTO lesion in each CTO patient (lesion-level analysis). RESULTS: Among the 216 patients, 32 (14.8%) had a CTO lesion on ICA. In patient-level analysis, no significant differences in clinical characteristics were found, whereas the coronary CTA culprit lesions of the CTO group had a smaller minimal lumen diameter (MLD) with more adverse plaque characteristics. In lesion-level analysis, future CTO lesions had a smaller MLD, a smaller reference segment diameter (RD), and longer lesion length. These lesions were more likely to be noncalcified plaques with a noneccentric cross-sectional distribution, and had a higher remodeling index, lower mean plaque attenuation (MPA), and more napkin-ring signs. In multivariate analysis and receiver-operating characteristic curve analysis, MLD of <2.0 mm, RD of <3.2 mm, and MPA of <50 Hounsfield units were independent predictors of future CTO lesions. The risk of CTO development in lesions with triple risk factors was 14-fold higher than that of the lesions with no risk factors. CONCLUSIONS: Lesions that progressed to CTO had more severe baseline coronary CTA features than non-CTO lesions. A small MLD, small RD, and low MPA were independent predictors of progression to CTO.

Female sex, central lymph node metastasis and dissection are causes of globus symptom after thyroidectomy.

Globus symptoms are not uncommon after an uncomplicated thyroidectomy. However, their associated factors and etiology have not been investigated. We investigated the etiology and factors related to globus symptoms after thyroidectomy. The medical records of 289 patients who underwent thyroidectomy and completed a voice analysis, psychiatric screening, and voice-related questionnaires before and 1 month after the surgery were reviewed. Patients were excluded if they had globus symptoms before surgery or scored high on the psychiatric questionnaire. The selected patients were divided into two groups according to development of globus symptoms after surgery. Clinicopathological parameters and results of the voice analysis and voice-related questionnaires were compared between the two groups. A total of 157 patients were enrolled, and more than half (80/155, 51 %) showed development of globus symptoms 1 month after thyroidectomy. Female patients [hazard ratio (HR), 2.605; P = 0.010], patients who had central lymph node metastasis (HR, 3.533; P = 0.001), and patients who underwent central neck dissection (HR, 3.652; P = 0.014) had a higher probability of developing globus symptoms. Patients who developed globus symptoms scored higher on the voice-related questionnaire, and had a greater decrease in speaking fundamental frequency (P < 0.001). Globus symptoms developed after 1 month in more than half of patients who underwent thyroidectomy. Female sex and central lymph node metastasis and dissection increased the possibility of developing the symptoms.

A 14-week randomized, placebo-controlled, double-blind clinical trial to evaluate the efficacy and safety of ginseng polysaccharide (Y-75).

BACKGROUND: The Y-75 (Ginsan) acidic polysaccharide from Korean Panax ginseng has been shown to function as an immunomodulatory molecule. However, the efficacy of Y-75 has not been evaluated in clinical trial. METHODS: We verified Y-75 (6 g/day) for safety and immune efficacy in 72 healthy volunteers aged 50-75 years using a randomized, placebo-controlled, parallel, double-blind study. The activities of natural killer (NK) cells and peripheral blood phagocytes, as well as serum levels of monocyte-derived mediators, were assessed before and after administration for 8 and 14 weeks. This trial is registered at ClinicalTrials.gov (NCT02161198). RESULTS: Y-75 significantly enhanced NK cell cytotoxic activity by 35.2% and 40.2% from baseline after administration for 8 and 14 weeks, respectively. The phagocytic activity of peripheral blood cells was also significantly increased by 25.2% and 39.4% and serum level of TNF-α by 38.2% and 44.5% after treatment for 8 and 14 weeks, respectively. Differences in the efficacy of variables compared to the placebo group were also significant. Administration of Y-75 was well tolerated without treatment-related adverse events or alteration of complete blood cell count or blood chemistry over the entire study period. CONCLUSION: Y-75 was shown to be a safe and potentially effective natural alternative for enhancing immune function.

Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2.

We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX2(1-25). While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases.DOI: http://dx.doi.org/10.7554/eLife.02667.001.

Structure of the TRAF4 TRAF domain with a coiled-coil domain and its implications for the TRAF4 signalling pathway.

The TNF receptor-associated factor (TRAF) proteins are structurally similar scaffold proteins that mediate between members of the TNF receptor (TNFR) family and downstream effector molecules such as kinases in the immune signalling pathway. Seven TRAFs have been identified, including TRAF4, which is a unique member that participates in many ontogenic processes, including nerve-system development. TRAFs commonly contain the TRAF domain, which mediates interaction with target receptors and effectors. As a first step towards elucidating the molecular mechanisms of the TRAF4-mediated signalling pathway, the first crystal structure of the human TRAF4 TRAF domain with a coiled-coil domain is reported at 2.3 Šresolution.

Cefepime- Induced Non-Convulsive Status Epilepticus (NCSE).

Cefepime is a fourth-generation B-lactam cephalosporin, commonly used in immunosuppressed patients. Neurotoxicity, which present as nonconvulsive status epilepticus (NCSE), has been reported previously especially in adult patients with impaired renal function. We present a case of cefepime induced NCSE after recovering from acute renal failure. A 71-year-old woman was hospitalized for right lower lobe lobectomy after diagnosis of lung cancer. Although she had successful lobectomy, she underwent several post operative complication including operation site bleeding, acute renal failure, acute respiratory distress syndrome, and atypical pneumonia. Her renal failure was prerenal type after massive operation site bleeding, and continuous renal replacement therapy (CRRT) were started for renal replacement treatment. After 5 days of renal replacement therapy, her serum creatinine level was much improved from 2.7 mg/dL to 1.33 mg/dL. Cefepime renal dose were started, when atypical pneumonia became resistant to imipenem and vancomycin. After 5th day of cefepime use, the patient became stupor and developed one episode of brief generalized myoclonic seizure. Her electroencephalograph (EEG) revealed 2-3 Hz generalized sharp and with impression of NCSE, she was started on anti-epileptic treatment. Clinical symptoms improved 3 days after discontinuation of cefepime. She was than diagnosed with cefepime induced non convulsive status epilepticus. Anti-epileptic treatments were than discontinued uneventfully. Awareness of the potential neurotoxic clinical manifestations of various antibiotics and high degree of vigilance in critically ill patients is essential in identifying a potentially serious though reversible complication of antibiotic therapy.

Regulator of Calcineurin 1 Isoform 4 (RCAN1.4) Is Overexpressed in the Glomeruli of Diabetic Mice.

Calcineurin (CaN) is activated in diabetes and plays a role in glomerular hypertrophy and extracellular matrix (ECM) accumulation. Here, kidneys from diabetic model mice were investigated for the expression of the regulator of CaN 1 (RCAN1) isoform 4 (RCAN1.4) which had been shown to be transcriptionally upregulated by CaN activation. We found the increased immunoreactivity for RCAN1 in the glomerular cells of db/db mice and streptozotocin-induced diabetic mice. In concordance, the expression of RCAN1 protein and RCAN1.4 mRNA were elevated in the whole kidney sample from db/db mice. Interleukin-1ß (IL-1ß), tumor necrosis factor-α, and glycated albumin (AGE-BSA) were identified as inducers of RCAN1.4 in mesangial cells. Pretreatment of cyclosporine A blocked the increases of RCAN1.4 stimulated by IL-1ß or AGE-BSA, suggesting that activation of CaN is required for the RCAN1.4 induction. Stable transfection of RCAN1.4 in Mes-13 mesangial cells upregulated several factors relevant to ECM production and degradation. These results suggested that RCAN1.4 might act as a link between CaN activation and ECM turnover in diabetic nephropathy.

Bcr and Abr cooperate in negatively regulating acute inflammatory responses.

Bcr and Abr are GTPase-activating proteins for the small GTPase Rac. Both proteins are expressed in cells of the innate immune system, including neutrophils and macrophages. The function of Bcr has been linked to the negative regulation of neutrophil reactive oxygen species (ROS) production, but the function of Abr in the innate immune system was unknown. Here, we report that mice lacking both proteins are severely affected in two models of experimental endotoxemia, including exposure to Escherichia coli lipopolysaccharide and polymicrobial sepsis, with extensive microvascular leakage, resulting in severe pulmonary edema and hemorrhage. Additionally, in vivo-activated neutrophils of abr and bcr null mutant mice produced excessive tissue-damaging myeloperoxidase (MPO), elastase, and ROS. Moreover, the secretion of the tissue metalloproteinase MMP9 by monocytes and ROS by elicited macrophages was abnormally high. In comparison, ROS production from bone marrow monocytes was not significantly different from that of controls, and the exocytosis of neutrophil secondary and tertiary granule products, including lactoferrin, was normal. These data show that Abr and Bcr normally curb very specific functions of mature tissue innate immune cells, and that each protein has distinct as well as partly overlapping functions in the downregulation of inflammatory processes.

Upregulation of DSCR1 (RCAN1 or Adapt78) in the peri-infarct cortex after experimental stroke.

Down syndrome candidate region 1 (DSCR1; also known as RCAN1 or Adapt78) has been shown to be induced by calcium overload and oxidative stress which are included in the pathogenic hallmarks of the ischemic diseases. After ischemic stroke, inflammatory responses play an important role in the exacerbation of neuronal loss. In this study, we investigated the expression pattern of DSCR1 in the mouse cortex after transient middle cerebral artery occlusion (MCAO). Then, in vitro studies were taken to address whether inflammatory mediators could induce DSCR1. Male C57BL/6 mice were subjected to transient MCAO for 35 min and sacrificed at 6, 24, and 72 h after the reperfusion. The expression of DSCR1 began to increase in layer VI of the peri-infarct cortex at 24 h and was prominently enhanced at 72h after transient MCAO. Moreover, real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry showed that the induction of the DSCR1 isoform 4 (DSCR1-4) mRNA preceded the expression of the DSCR1 protein. In in vitro studies, tumor necrosis factor alpha and interleukin-1beta (IL-1beta) were found to induce strong upregulation of DSCR1-4 mRNA. Furthermore, western blot analysis revealed that overexpression of DSCR1-4 in SK-N-SH neuroblastoma cells attenuated IL-1beta-induced cyclooxygenase 2 and intercellular adhesion molecule 1 expression. These results demonstrate upregulation of DSCR1 in the mouse peri-infarct cortex following transient MCAO. In addition, our results suggest that inflammatory mediators such as TNFalpha and IL-1beta can induce DSCR1-4 transcription, which may be associated with the alleviation of inflammatory processes.

Activity of the Bcr GTPase-activating domain is regulated through direct protein/protein interaction with the Rho guanine nucleotide dissociation inhibitor.

The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by guanine nucleotide exchange factors, GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). Little is known about how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert Rac-GTP to Rac-GDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to Rac-GTP and catalyzes conversion of the bound GTP to GDP on Rac. The presence of activated Rac diminished the Bcr/RhoGDIalpha interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of Rac-GTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, indicating that binding of RhoGDIalpha and Rac-GTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates BcrGAP activity.

Femoral bioabsorbable cross-pin fixation in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to retrospectively evaluate clinical results after arthroscopic anterior cruciate ligament (ACL) reconstruction by use of hamstring autograft with femoral fixation via 2 bioabsorbable cross pins (Rigidfix system; DePuy Mitek, Raynham, MA). METHODS: We evaluated the results of 117 knees (117 patients) that had been treated with arthroscopic ACL reconstruction by use of hamstring autograft with femoral fixation via 2 bioabsorbable cross pins from September 2001 to November 2002. The mean follow-up period was 26.9 months (range, 25 to 32 months). Patients were evaluated by the Lachman test, pivot-shift test, KT-2000 arthrometer (MEDmetric, San Diego, CA) test, Lysholm score, and International Knee Documentation Committee grade at the preoperative and follow-up examinations. Of the knees, 74 were assessed by second-look arthroscopy. RESULTS: The mean Lysholm score was 72.6 (range, 51 to 86) preoperatively and 93.7 (range, 71 to 99) postoperatively. On the basis of the final International Knee Documentation Committee grade, 75 knees were normal (A), 36 were nearly normal (B), 6 were abnormal (C), and none were severely abnormal (D). KT-2000 arthrometer analysis revealed that 112 knees (95.7%) were grade A or B, with a median laxity of 1.3 mm (range, 1 to 6 mm) at final follow-up. Of the 74 knees that were assessed by second-look arthroscopy, 52 had preserved good tension, 22 had some laxity, and none had graft failure or rupture. CONCLUSIONS: ACL reconstruction via 4-strand hamstring autograft with femoral fixation by use of 2 bioabsorbable cross pins eliminated anterior tibial translation in 93.1% of patients at a mean follow-up of 26.9 months. The bioabsorbable cross-pin femoral fixation method via hamstring tendon autograft can be effective, useful, and reproducible. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Regional differences in the neuroprotective effect of minocycline in a mouse model of global forebrain ischemia.

We investigated the effect of minocycline on neuronal damage in the hippocampus and striatum in a mouse model of transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral occlusion of the common carotid artery (BCCAO) for 30 min. Minocycline (90 mg/kg, i.p., qd) or saline was injected immediately after BCCAO and daily for the next two days (45 mg/kg, i.p., bid). In order to reduce the variability in ischemic neuronal damage, we applied selection criteria based on regional cerebral blood flow (rCBF), evaluated using laser Doppler flowmetry, and the plasticity of the posterior communicating artery (PcomA), evaluated using India ink solution. In animals with rCBF that was less than 15% of the baseline value and with a smaller PcomA, of diameter less than one-third that of the basilar artery, we consistently observed neuronal damage in the striatum and hippocampal subfields, including medial CA1, CA2, and CA4. When the effect of minocycline was assessed with cresyl violet staining, neuronal damage in the medial part of the CA1 subfield and the striatum was found to be significantly attenuated, although minocycline did not protect against neuronal damage in the remaining hippocampal subfields. Immunohistochemistry for NeuN, adenosine A1 receptor, and SCIP/Oct-6 confirmed the region-specific effect of minocycline in the hippocampus. In summary, our results suggest that minocycline protects neurons against global forebrain ischemia in a subregion-specific manner.

Abr and Bcr, two homologous Rac GTPase-activating proteins, control multiple cellular functions of murine macrophages.

Small GTPases of the Rho family are key regulators of phagocytic leukocyte function. Abr and Bcr are homologous, multidomain proteins. Their C-terminal domain has GTPase-activating protein (GAP) activity that, in vitro, is specific for Rac and Cdc42. To address the in vivo relevance of these entire proteins, of which little is known, the current study examined the effect of the genetic ablation of Abr and Bcr in murine macrophages. The concomitant loss of Abr and Bcr induced multiple alterations of macrophage cellular behavior known to be under the control of Rac. Macrophages lacking both Abr and Bcr exhibited an atypical, elongated morphology that was reproduced by the ectopic expression of GAP domain mutant Abr and Bcr in a macrophage cell line and of constitutively active Rac in primary macrophages. A robust increase in colony-stimulating factor 1 (CSF-1)-directed motility was observed in macrophages deficient for both proteins and, in response to CSF-1 stimulation, Abr and Bcr transiently translocated to the plasma membrane. Phagocytosis of opsonized particles was also increased in macrophages lacking both proteins and correlated with sustained Rac activation. Bcr and Abr GAP mutant proteins localized around phagosomes and induced distinct phagocytic cup formation. These results identify Abr and Bcr as the only GAPs to date that specifically negatively regulate Rac function in vivo in primary macrophages.

Down syndrome candidate region 1 increases the stability of the IkappaBalpha protein: implications for its anti-inflammatory effects.

Down syndrome candidate region 1 (DSCR1), an endogenous inhibitor of calcineurin, inhibits the expression of genes involved in the inflammatory response. To elucidate the molecular basis of these anti-inflammatory effects, we analyzed the role of DSCR1 in the regulation of NF-kappaB transactivation using glioblastoma cells stably transfected with DSCR1.4 or its truncation mutants (DSCR1.4-(1-133) and DSCR1.4-(134-197)). Overexpression of DSCR1.4 significantly attenuated the induction of cyclooxygenase-2 (COX-2) expression by phorbol 12-myristate 13-acetate (PMA) via a calcineurin-independent mechanism. Experiments using inhibitors of the signaling molecules for NF-kappaB activation showed that NF-kappaB is responsible for the induction of COX-2. Full-length and truncated DSCR1.4 decreased the steady-state activity of NF-kappaB as well as PMA-induced activation of NF-kappaB, which correlated with attenuation of COX-2 induction. DSCR1.4 did not affect the PMA-stimulated phosphorylation or degradation kinetics of IkappaBalpha; however, DSCR1.4 significantly decreased the basal turnover rate of IkappaBalpha and consequently up-regulated its steady-state level. In the same context, knockdown of endogenous DSCR1.4 increased the turnover rate of IkappaBalpha as well as COX-2 induction. These results suggest that DSCR1 attenuates NF-kappaB-mediated transcriptional activation by stabilizing its inhibitory protein, IkappaBalpha.