Epigenetic silencing of the human NOS2 gene: rethinking the role of nitric oxide in human macrophage inflammatory responses.

Macrophages, including alveolar macrophages, are primary phagocytic cells of the innate immune system. Many studies of macrophages and inflammation have been done in mouse models, in which inducible NO synthase (NOS2) and NO are important components of the inflammatory response. Human macrophages, in contrast to mouse macrophages, express little detectable NOS2 and generate little NO in response to potent inflammatory stimuli. The human NOS2 gene is highly methylated around the NOS2 transcription start site. In contrast, mouse macrophages contain unmethylated cytosine-phosphate-guanine (CpG) dinucleotides proximal to the NOS2 transcription start site. Further analysis of chromatin accessibility and histone modifications demonstrated a closed conformation at the human NOS2 locus and an open conformation at the murine NOS2 locus. In examining the potential for CpG demethylation at the NOS2 locus, we found that the human NOS2 gene was resistant to the effects of demethylation agents both in vitro and in vivo. Our data demonstrate that epigenetic modifications in human macrophages are associated with CpG methylation, chromatin compaction, and histone modifications that effectively silence the NOS2 gene. Taken together, our findings suggest there are significant and underappreciated differences in how murine and human macrophages respond to inflammatory stimuli.

Rituximab infusion induces NK activation in lymphoma patients with the high-affinity CD16 polymorphism.

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity involving FcγRIIIa (CD16) likely contributes to the clinical efficacy of rituximab. To assess the in vivo effects of CD16 polymorphisms on rituximab-induced NK activation, blood was evaluated before and 4 hours after initiation of the initial dose of rituximab in 21 lymphoma subjects. Rituximab induced NK activation and a drop in circulating NK-cell percentage in subjects with the high-affinity [158(VF/VV)] but not the low-affinity [158(FF)] CD16 polymorphism. There was no correlation between NK-cell activation or NK-cell percentage and polymorphisms in CD32A, C1q, or CH50. We conclude that NK activation occurs within 4 hours of rituximab infusion in subjects with the high-affinity CD16 polymorphism but not those with the low-affinity CD16 polymorphism. This finding may help explain the superior clinical outcome seen in the subset of high-affinity CD16 polymorphism lymphoma patients treated with single-agent rituximab.

Correlation of MTDH/AEG-1 and HOTAIR Expression with Metastasis and Response to Treatment in Sarcoma Patients.

BACKGROUND: Chemoresistance and metastasis are the main reasons for the failure of current treatments with sarcoma patients. Novel biomarkers are required to predict metastasis and response to treatment. The oncogene MTDH/AEG1 and the long noncoding RNA (lincRNA) HOTAIR are two novel factors involved in drug resistance and metastasis in various types of solid tumors. However, the correlation between MTDH/AEG-1 and HOTAIR expression with metastasis and drug resistance in sarcoma is unknown. METHODS: Expression of MTDH protein or HOTAIR was detected by Western blotting or qRT-PCR, respectively, in primary and metastatic sarcoma patient tissue samples. RESULTS: High individual or co-expression of MTDH/AEG1 and HOTAIR was observed in three of four primary and six of eight metastatic sarcoma patient tumor samples. High level expression of both of MTDH/AEG1 and HOTAIR in the primary tumor correlated with a likelihood to metastasize. MTDH expression was lower in samples pre-treated with irradiation and/or chemotherapy as compared to those that had not been treated. HOTAIR expression seemed to correlate with the percent necrosis seen in different sarcoma samples. CONCLUSIONS: High levels of both MTDH/AEG-1 and HOTAIR in primary sarcoma are correlated with a high probability of metastasis. By contrast, reduced expression of both MTDH/AEG-1 and HOTAIR is correlated with a good response to treatment in terms of necrosis, suggesting that levels of MTDH and HOTAIR are potential biomarkers for treatment efficacy. Whether we can predict disease progression in sarcoma remains to be seen. Additional study is needed to better define the best clinical application of MTDH/AEG-1 and HOTAIR expression with metastasis and outcome.

[Not Available].

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently being investigated as a therapeutic agent for a variety of malignancies, as it triggers apoptosis specifically in transformed cells. However, TRAIL use as a stand alone therapeutic is hampered by the fact that many primary tumor cells are resistant to TRAIL-mediated apoptosis. Here, we investigated the extent to which pretreatment of TRAIL-resistant primary B-cell chronic lymphocytic leukemia (B-CLL) cells with histone deacetylase inhibitors (HDACis) could render them susceptible to killing by TRAIL. We found that HDAC inhibition in B-CLL cells led to increased TRAIL receptor expression, increased caspase activation, decreased expression of antiapoptotic regulators such as Bcl-2, and ultimately, enhanced TRAIL-induced apoptosis. Importantly, untransformed peripheral blood mononuclear cells remained largely resistant to TRAIL, even in the presence of HDACis. These results suggest that combination therapies using HDAC inhibition and TRAIL could prove beneficial for the treatment of B-CLL.

In vivo suppression of polyglutamine neurotoxicity by C-terminus of Hsp70-interacting protein (CHIP) supports an aggregation model of pathogenesis.

Perturbations in neuronal protein homeostasis likely contribute to disease pathogenesis in polyglutamine (polyQ) neurodegenerative disorders. Here we provide evidence that the co-chaperone and ubiquitin ligase, CHIP (C-terminus of Hsp70-interacting protein), is a central component to the homeostatic mechanisms countering toxic polyQ proteins in the brain. Genetic reduction or elimination of CHIP accelerates disease in transgenic mice expressing polyQ-expanded ataxin-3, the disease protein in Spinocerebellar Ataxia Type 3 (SCA3). In parallel, CHIP reduction markedly increases the level of ataxin-3 microaggregates, which partition in the soluble fraction of brain lysates yet are resistant to dissociation with denaturing detergent, and which precede the appearance of inclusions. The level of microaggregates in the CNS, but not of ataxin-3 monomer, correlates with disease severity. Additional cell-based studies suggest that either of two quality control ubiquitin ligases, CHIP or E4B, can reduce steady state levels of expanded, but not wild-type, ataxin-3. Our results support an aggregation model of polyQ disease pathogenesis in which ataxin-3 microaggregates are a neurotoxic species, and suggest that enhancing CHIP activity is a possible route to therapy for SCA3 and other polyQ diseases.

Selective cochlear degeneration in mice lacking the F-box protein, Fbx2, a glycoprotein-specific ubiquitin ligase subunit.

Little is known about the role of protein quality control in the inner ear. We now report selective cochlear degeneration in mice deficient in Fbx2, a ubiquitin ligase F-box protein with specificity for high-mannose glycoproteins (Yoshida et al., 2002). Originally described as a brain-enriched protein (Erhardt et al., 1998), Fbx2 is also highly expressed in the organ of Corti, in which it has been called organ of Corti protein 1 (Thalmann et al., 1997). Mice with targeted deletion of Fbxo2 develop age-related hearing loss beginning at 2 months. Cellular degeneration begins in the epithelial support cells of the organ of Corti and is accompanied by changes in cellular membrane integrity and early increases in connexin 26, a cochlear gap junction protein previously shown to interact with Fbx2 (Henzl et al., 2004). Progressive degeneration includes hair cells and the spiral ganglion, but the brain itself is spared despite widespread CNS expression of Fbx2. Cochlear Fbx2 binds Skp1, the common binding partner for F-box proteins, and is an unusually abundant inner ear protein. Whereas cochlear Skp1 levels fall in parallel with the loss of Fbx2, other components of the canonical SCF (Skp1, Cullin1, F-box, Rbx1) ubiquitin ligase complex remain unchanged and show little if any complex formation with Fbx2/Skp1, suggesting that cochlear Fbx2 and Skp1 form a novel, heterodimeric complex. Our findings demonstrate that components of protein quality control are essential for inner ear homeostasis and implicate Fbx2 and Skp1 as potential genetic modifiers in age-related hearing loss.

[A case report. Emphysematous cystitis--a rare complication of urinary tract infection]. / Fallbeskrivning. Emfysematös cystit--sällsynt komplikation till urinvägsinfektion.

An elderly woman with poorly controlled diabetes entered the casualty department suffering from abdominal pain. Clinical and radiological examination revealed intramural and intraluminal gas in the urinary bladder, suggesting the diagnosis as emphysematous cystitis. It is a rare disorder usually caused by aerobic and not anaerobic microbes. The causal agent is often, as in this case, Escherichia coli.

Horseshoe kidney with a circumcaval ureter.

Horseshoe kidney accompanied by a totally circumcaval ureter has rarely been reported in the same patient. However, both entities are well recognized as being uncommon anomalies of the genitourinary system. Variability in the normal course of the ureter makes it difficult to differentiate a normal variant from a congenital anomaly or a deviation due to other factors such as inflammation or cancer. We present a case of simultaneous horseshoe kidney and a right ureter that ran in a circular fashion (360 degrees) around the inferior vena cava. Contrast-enhanced CT showed that the ureter passed posterior and medial to the inferior vena cava, providing the definitive diagnosis.

Emphysematous cystitis.

An elderly woman with poorly controlled diabetes entered the casualty department suffering from abdominal pain. Clinical and radiological examinations revealed intramural and intraluminal gas in the urinary bladder, suggesting the diagnosis as emphysematous cystitis, a rare disorder usually caused by aerobic and not anaerobic microbes. The causal agent is often, as in this case, Escherichia coli.

Optimizing levodopa pharmacokinetics: intestinal infusion versus oral sustained-release tablets.

Continuous duodenal infusion of carbidopa/levodopa has been shown to control motor fluctuations in advanced Parkinson's disease (PD). The authors compared the pharmacokinetics of levodopa and 3-O-methyldopa in patients with advanced PD after administration of an oral sustained-release levodopa preparation and after continuous intestinal levodopa infusion with a new formulation as a gel suspension. A randomized crossover trial was carried out in 12 patients. Carbidopa/levodopa was administered as an oral sustained-release tablet and by nasoduodenal continuous infusion for 3-week periods for each treatment. Plasma levodopa concentrations and motor performance were evaluated every 30 minutes during 3 test days of each treatment period. The average intraindividual coefficient of variation for the plasma levodopa concentrations after oral therapy was 34% and was significantly lower (14%, p < 0.01) during continuous infusion. Hourly video evaluations showed a significant increase in ON time during infusion and a significant decrease in OFF time and dyskinesia. Continuous intraduodenal delivery of a new carbidopa/levodopa formulation offers a means for markedly improved control of motor fluctuations in late stages of PD.

The effect of ketoconazole on the in vivo intestinal permeability of fexofenadine using a regional perfusion technique.

AIMS: To investigate whether the drug-drug interaction between fexofenadine and ketoconazole is localized to efflux transport proteins of the small intestine, and to determine and classify the effective jejunal permeability (Peff) of fexofenadine according to the Biopharmaceutics Classification System (BCS). METHODS: Two separate jejunal perfusion experiments were performed using the Loc-I-Gut technique in eight healthy volunteers. During treatment 1 (T1), we investigated the acute effect of ketoconazole on the Peff and plasma pharmacokinetics of fexofenadine. In treatment 2 (T2) we examined the effect of oral pretreatment with ketoconazole (200 mg daily for 5 days) on the same absorption parameters. Each experiment was divided into two periods of 100 min and the jejunal segment was perfused with 93 micro m fexofenadine during both periods. In period 2 of each treatment, fexofenadine was coadministered with 94 micro m ketoconazole. The concentrations of fexofenadine in intestinal perfusate and plasma were measured by liquid chromatography with mass detection. RESULTS: During T1, the mean (+/- s.d.) Peff of fexofenadine was low according to the BCS (0.11 +/- 0.11 and 0.04 +/- 0.13 x 10(-4) cm s(-1) in periods 1 and 2, respectively), and the coadministration of ketoconazole in period 2 had no significant acute effect on Peff (95% confidence interval (CI) on the difference -0.37, 0.51). After pretreatment with ketoconazole (T2), the jejunal Peff of fexofenadine increased to 0.29 +/- 0.47 and 0.22 +/- 0.31 x 10-4 cm s(-1) in both periods 1 and 2, respectively, but the change was not statistically significant when compared with T1 (95% CI on the difference -0.62, 0.27 for T1 0-100 min vs T2 0-100 min; -0.54, 0.34 for T1 0-100 min vs T2 100-200 min). Fexofenadine plasma AUC from 0-100 mg showed no significant difference after pretreatment with ketoconazole (55 +/- 101 and 51 +/- 33 micro g ml(-1) min(-1) respectively; 95% CI on the difference -108, 115). Total plasma AUC (0-720 min) was 318 +/- 426 and 426 +/- 232 ng ml(-1) min in T1 and T2, respectively (95% CI on the difference -622, 405). CONCLUSIONS: No significant effect of acute coadministration or pretreatment with ketoconazole on the in vivo intestinal absorption of fexofenadine was detected in this study.