Mutations in the nucleophosmin gene (NPM1(mut)) are one of the most frequent molecular alterations in AML and immune responses might contribute to the favorable prognosis of AML patients with NPM1(mut). In this work, we could demonstrate both CD4+ and CD8+ T cell responses against NPM1(mut). Ten peptides derived from NPM1(wt) and NPM1(mut) were subjected to ELISpot analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against most interesting epitopes were performed and chromium release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4+ T cells in NPM1 immunogenicity. Two epitopes (#1,#3) derived from NPM1(mut) induced CD8+ T cell responses. 33% of the NPM1(mut) AML patients showed immune responses against #1 and 44% against #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4+ T cells is crucial. Therefore, overlapping peptides (OL) were analyzed in ELISpot assays and OL8 showed favorable results to activate both CD8+ and CD4+ T cells. Taken together, NPM1(mut) induces specific T cell responses of CD4+ and CD8+ T cells and thus presents a promising target for specific immunotherapies in AML.

BACKGROUND Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKβ is one of the subtypes of the DGK family and regulates many intracellular signaling pathways in the central nervous system. Previously, we demonstrated that DGKβ knockout (KO) mice showed various dysfunctions of higher brain function, such as cognitive impairment (with lower spine density), hyperactivity, reduced anxiety, and careless behavior. In the present study, we conducted further tests on DGKβ KO mice in order to investigate the function of DGKβ in the central nervous system, especially in the pathophysiology of attention deficit hyperactivity disorder (ADHD). METHODOLOGY/PRINCIPAL FINDINGS DGKβ KO mice showed attention-deficit behavior in the object-based attention test and it was ameliorated by methylphenidate (MPH, 30 mg/kg, i.p.). In the open field test, DGKβ KO mice displayed a decreased response to the locomotor stimulating effects of MPH (30 mg/kg, i.p.), but showed a similar response to an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.3 mg/kg, i.p.), when compared to WT mice. Examination of the phosphorylation of extracellular signal-regulated kinase (ERK), which is involved in regulation of locomotor activity, indicated that ERK1/2 activation induced by MPH treatment was defective in the striatum of DGKβ KO mice. CONCLUSIONS/SIGNIFICANCE These findings suggest that DGKβ KO mice showed attention-deficit and hyperactive phenotype, similar to ADHD. Furthermore, the hyporesponsiveness of DGKβ KO mice to MPH was due to dysregulation of ERK phosphorylation, and that DGKβ has a pivotal involvement in ERK regulation in the striatum.

BACKGROUND: Symptoms of choledochal cysts sometimes persist or become exacerbated. As preoperative management for patients with these cysts, we prospectively employed endoscopic drainage, based on the theory that protein plugs cause symptoms by obstructing the pancreatobiliary ducts. METHODS: Children with choledochal cysts underwent endoscopic retrograde cholangiopancreatography (ERCP). When ERCP showed compaction with filling defects in patients with persistent or worsening symptoms (study patients), the placement of a short biliary stent tube was attempted for drainage. The clinical and ERCP findings of the study patients were compared with those of patients who were asymptomatic at ERCP (asymptomatic patients). RESULTS: There were 13 study patients (median age 2.9 years) and 41 asymptomatic patients (4.7 years) enrolled in the study between August 2005 and February 2011. Study patients more frequently had jaundice and elevated transaminase levels. ERCP showed that all study patients had obstruction or compacted filling defects in the common channel or the narrow segment distal to the cyst. Insertion of a stent tube was successful in 11 patients. Symptoms were relieved soon after biliary drainage. Surgery revealed that the obstructing materials were protein plugs, except in one case, which involved fatty acid calcium stones. CONCLUSIONS: These results support the protein plug theory. Endoscopic short-tube stenting is adequate and effective as preoperative management.

Calpains, a family of Ca(2+)-dependent cytosolic cysteine proteases, can modulate their substrates' structure and function through limited proteolytic activity. In the human genome, there are 15 calpain genes. The most-studied calpains, referred to as conventional calpains, are ubiquitous. While genetic studies in mice have improved our understanding about the conventional calpains' physiological functions, especially those essential for mammalian life as in embryogenesis, many reports have pointed to overactivated conventional calpains as an exacerbating factor in pathophysiological conditions such as cardiovascular diseases and muscular dystrophies. For treatment of these diseases, calpain inhibitors have always been considered as drug targets. Recent studies have introduced another aspect of calpains, that calpain activity is required to protect the heart and skeletal muscle against stress. This review summarizes the functions and regulation of calpains, focusing on the relevance of calpains to cardiovascular disease.

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Because neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination, and proteasome processing for cross-presentation. Using anti-PR1/human leukocyte antigen-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, whereas DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classic major histocompatibility class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia.

A 32-year-old man was admitted to our hospital because of fever, headache, and loss of consciousness. Four days before admission, he had had difficulty speaking. On the day of admission, his colleague had found him to be unconscious and lying on his back. He was admitted to our hospital. The temperature at the eardrum was 35.2°C. Neurologic evaluation was negative. Computed tomography (CT) scan of the brain showed slight ventricular enlargement bilaterally. An X-ray film of the chest showed no abnormality. On the second hospital day, neck stiffness was noted. The cerebrospinal fluid (CSF) contained 870 white cells/μl, most of which were neutrophils; the glucose level in the CSF was 10 mg/dl, and the protein level was 140 mg/dl. Stained smears of the CSF, including Gram staining and India-ink preparations, disclosed no microorganisms. Capsular antigen tests for several bacteria were negative. Antimicrobial agents were started. However, by changing the microscope focus slightly while viewing Gram stains of the CSF, we could see brightened and Gram-positive bacilli that had been phagocytosed by neutrophils. This finding suggested the presence of Mycobacterium tuberculosis. Ziehl-Neelsen staining of the CSF and gastric juice revealed anti-acid bacilli. Polymerase chain reaction for M. tuberculosis in the gastric juice was positive. This case showed that Gram staining could be useful as an initial adjunct for the diagnosis of tuberculous meningitis, particularly when the CSF shows predominantly neutrocytic pleocytosis, but no other evidence of bacterial meningitis.

We examined the effect of the oral administration of β-D-glucan derived from Aureobasidium pullulans ADK-34 (AP-FBG) on Candida albicans or methicillin-resistant Staphylococcus aureus (MRSA) infection in immunosuppressed mice. Mice pretreated with cyclophosphamide (CY) were intraperitoneally administered AP-FBG for 4 days and then infected with 6×10(4) C. albicans cells. In a preliminary experiment, the survival time of the Candida-infected mice treated with AP-FBG was clearly prolonged. Similarly, the effect of the oral administration of AP-FBG was examined. Mice were orally given 2.5% AP-FBG in feed for 42 days from 14 days prior to 2×10(4) C. albicans cells infection. The survival time of mice treated with AP-FBG was significantly prolonged and the viable cell count in the kidneys of the survivors was significantly decreased at 30 days after infection. The effects of the oral administration of AP-FBG on intestinal MRSA infection were also examined. Mice were given 2.5% AP-FBG orally in feed for 30 days before and after oral MRSA infection and treated with CY 12 days after the infection. The number of viable MRSA cells or the IgA production in feces did not significantly change, while AP-FBG administration seemed to relieve temporally the loss of body weight of mice.Conclusions: These results suggest that oral pre-administration of AP-FBG promoted resistance of CY-treated mice to C. albicans and lessened the weight reduction of CY-mice infected by MRSA.

The aim of the present study was to assess changes of cell membrane antigens on neutrophils in septic patients. Expression levels of neutrophil membrane antigens were measured employing a FACS calibur flow cytometer with several fluorescence-labeled monoclonal antibodies. Expression levels of the CD14 antigen were higher in patients with sepsis than in healthy individuals. In particular, the expression levels of CD14 increased in patients complicated by septic shock. Expression levels of TLR-4 were higher in patients with sepsis or septic shock than in healthy individuals. Expression levels of CD11b and CD16 were lower in patients with sepsis or septic shock than in healthy individuals and were even lower in those complicated by septic shock. Expression levels of neutrophil membrane antigens in patients with sepsis markedly changed in the acute phase. However, these levels tended to return to those of healthy individuals in the convalescing phase. Analyses of the surface antigens on neutrophils strongly involved in biological defense or tissue injury are informative for understanding the pathology of sepsis and for conducting therapy targeting neutrophils in the future.

The treatment of psoriasis has undergone a revolution with the advent of biologic therapies including infliximab, etanercept, adalimumab, efalizumab, golimumab, certolizumab, alefacept, secukinumab, abatacept, and ustekinumab. These medications are designed to target specific components of the immune system and are a major technological advancement over traditional immunosuppressive medications. Herein, we present a comprehensive, unbiased comparison of these medications focusing on their differences. For example, TNF antagonists can differ in the way they are dissolved and administered, the effector molecules they can bind, serum peak and trough levels, the types of intracellular signals they can induce, the in vivo complexes that they can form, their protein structure, and their incidence and timing of rare adverse events, among other things. A critical review of the clinical studies that have tested the efficacy of these molecules is also presented including head-to-head comparison trials. The safety of biologics in terms of their long-term adverse events is discussed, as is their use in different types of psoriasis and in different patient populations. Finally, all anti-TNF agents have been associated with a variety of serious and "routine" opportunistic infections, particularly tuberculosis. For this reason, anti-tuberculosis testing both prior to the initiation of a biologic therapy and annually during treatment is pertinent. The uses and limitations of both the tuberculin skin test (TST) and QuantiFeron®-TB Gold (QFT) are discussed, as is the care of patients who present with latent tuberculosis infection prior to the initiation of biologic therapy. Recommendations for tuberculosis monitoring are provided.

We report on single electron pumping via a tunable number of individual donors. We use a device that essentially consists of a silicon nanowire with local arsenic implantation between a set of fine gates. A temperature-dependent characterization of the pumped current allows us to extract the ionization energy of a single arsenic donor. We observe the ionization energy to be tunable by the gate electric field over a large range of energies.