Binuclear copper(II) complexes of tolfenamic: synthesis, crystal structure, spectroscopy and superoxide dismutase activity

The synthesis and characterization of copper(II) complexes with a potent non-steroidal anti-inflammatory drug, tolfenamic acid, Htolf, with formula [Cu(tolf)(2)L](2) (where L is H(2)O or DMF, N,N-dimethylformamide) were investigated. The crystal and molecular structure of [Cu(tolf)(2)(DMF)](2) was reported. Crystallographic data are as follows: monoclinic system, space group P2(1)/n with cell constants a=9.068(2) A, b=14.514(3) A, c=22.826(4) A, V=2948.9(10) A(3) and Z=2. The crystal structure consists of binuclear, quadruply bridged neutral molecule with a Cu-Cu bond length of 2.6075(19) A. The complex is self-assembled via C-H-pi intermolecular stacking interactions. Spectroscopic and electrochemical studies were reported. The superoxide dismutase activity is measured and compared with those of superoxide dismutase enzyme, SOD, the free ligand and related copper complexes with non-steroidal anti-inflammatory drugs, NSAIDs. IC(50) value was measured by the Fridovich test (1.97+/-0.17 microM), which showed that [Cu(tolf)(2)L](2) is a good superoxide scavenger.     

Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

Economic Recession and Emergence of an HIV-1 Outbreak among Drug Injectors in Athens Metropolitan Area: A Longitudinal Study

Background

During 2011, a dramatic increase (1600%) of reported HIV-1 infections among injecting drug users (IDUs) was noted in Athens, Greece. We herein assess the potential causal pathways associated with this outbreak.

Methods

Our study employed high resolution HIV-1 phylogenetic and phylogeographic analyses. We examined also longitudinal data of ecological variables such as the annual growth of gross domestic product (GDP) of Greece in association with HIV-1 and HCV sentinel prevalence in IDUs, unemployment and homelessness rates and HIV transmission networks in Athens IDUs before and during economic recession (2008–2012).

Results

IDU isolates sampled in 2011 and 2012 suggested transmission networks in 94.6% and 92.7% of the cases in striking contrast with the sporadic networking (5%) during 1998–2009. The geographic origin of most HIV-1 isolates was consistent with the recently documented migratory waves in Greece. The decline in GDP was inversely correlated with annual prevalence rates of HIV and HCV and with unemployment and homelessness rates in IDUs (all p<0.001). The slope of anti-HCV prevalence in the sentinel populations of IDUs and in “new” drug injectors was found 120 and 1.9-fold (p = 0.007, p = 0.08 respectively) higher in 2008–2012 (economic recession) compared with 2002–2006. The median (25th, 75th) size of transmission networks were 34 (12, 58) and 2 (2, 2) (p = 0.057) in 2008–2012 and 1998–2007, respectively. The coverage of harm reduction services was low throughout the study period.

Conclusions

Scaling-up harm reduction services and addressing social and structural factors related to the current economic crisis should be urgently considered in environments where HIV-1 outbreaks may occur.     

Low Dose Infliximab for Prevention of Postoperative Recurrence of Crohn’s Disease: Long Term Follow-Up and Impact of Infliximab Trough Levels and Antibodies to Infliximab

Objective

In patients with postoperative recurrence of Crohn’s disease endoscopic and clinical remission can be maintained for up to 1 year with low infliximab doses (3 mg/Kg). However, in theory low-dose infliximab treated patients could develop subtherapeutic trough levels, infiximab antibodies, and might loose response to therapy. To verify this hypothesis infliximab pharmacokinetics and clinical/endoscopic response were checked in a group of patients treated in the long term with low infliximab doses.

Design

Infliximab antibodies, infliximab levels, highly-sensitive CRP and fecal calprotectin were measured during the 8-week interval in 5 consecutive patients in clinical (Crohn’s Disease Activity Index < 150) and endoscopic (Rutgeerts scores 0–1) remission after one year of therapy with infliximab 3 mg/Kg. For comparison with reported standards, infliximab pharmacokinetics and inflammatory parameters were also tested in 6 Crohn’s disease patients who did not undergo surgery and who were in clinical remission while on infliximab 5 mg/Kg. Patients on low infliximab dose also underwent colonoscopy after 18 additional months of therapy.

Results

Highly sensitive CRP and fecal calprotectin increased in all patients during the 8-week interval. Infliximab trough levels were lower in patients treated with the low dose compared to controls (mean±SE: 2.0±0.3 vs 4.75±0.83 μg/mL respectively p<0.05). Infliximab antibodies were present in two of the subjects treated with low infliximab dose and in none of the controls. However, in low dose-treated patients after 18 additional months of therapy endoscopy continued to show mucosal remission and none of them developed clinical recurrence or side effects.

Conclusions

Patients treated with low infliximab doses had lower trough levels compared to patients treated with 5 mg/Kg and some developed antibodies to infliximab. However, low infliximab doses sustained clinical and endoscopic remission for a total of 30 months of treatment.     

Complex regulation of the TRPM8 cold receptor channel: role of arachidonic acid release following M3 muscarinic receptor stimulation

Cold/menthol-activated TRPM8 (transient receptor potential channel melastatin member 8) is primarily expressed in sensory neurons, where it constitutes the principal receptor of environmental innocuous cold. TRPM8 has been shown to be regulated by multiple influences such as phosphorylation, pH, Ca(2+), and lipid messengers. One such messenger is arachidonic acid (AA), which has been shown to inhibit TRPM8 channel activity. However, the physiological pathways mediating the inhibitory effect of AA on TRPM8 still remain unknown. Here, we demonstrate that TRPM8 is regulated via M3 muscarinic acetylcholine receptor-coupled signaling cascade based on the activation of cytosolic phospholipase A2 (cPLA2) and cPLA2-catalyzed derivation of AA. Stimulation of M3 receptors heterologously co-expressed with TRPM8 in HEK-293 cells by nonselective muscarinic agonist, oxotremorine methiodide (Oxo-M), caused inhibition of TRPM8-mediated membrane current, which could be mimicked by AA and antagonized by pharmacological or siRNA-mediated cPLA2 silencing. Our results demonstrate the intracellular functional link between M3 receptor and TRPM8 channel via cPLA2/AA and suggest a novel physiological mechanism of arachidonate-mediated regulation of TRPM8 channel activity through muscarinic receptors. We also summarize the existing TRPM8 regulations and discuss their physiological and pathological significance.