Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination.     

Benfotiamine Attenuates Inflammatory Response in LPS Stimulated BV-2 Microglia

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.     

Urinary Biomarkers at Early ADPKD Disease Stage

Background

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by a decline in renal function at late disease stage when the majority of functional renal parenchyma is replaced by cystic tissue. Thus, kidney function, assessed by estimated glomerular filtration rate (eGFR) does not well represent disease burden in early disease. Here, we investigated various urinary markers for tubular injury and their association with disease burden in ADPKD patients at early disease course.

Methods

ADPKD patients between 18 and 40 years with an eGFR greater or equal to 70 ml per min per 1.73m² were eligible for this cross-sectional study. Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL), Kidney Injury Molecule-1 (KIM-1), and Uromodulin (UMOD) were investigated by Enzyme-Linked Immunosorbent Assay. Clara Cell Protein 16 (CC16) was investigated by Latex Immuno Assay. Cryoscopy was performed to assess urine osmolality and Urinary Albumin-to-Creatinine Ratio (UACR) was calculated. The association and the predictive properties of the markers on eGFR and height adjusted total kidney volume (htTKV) was evaluated using multiple regression analysis, incorporating different control variables for adjustment. Internal bootstrapping validated the obtained results.

Results

In 139 ADPKD patients (age 31 ±7 years, mean eGFR of 93 ± 19 ml per min per 1.73 m²) the total kidney volume was negatively correlated with eGFR and UMOD and positive associated with age, UACR, KIM-1 and urine osmolality after adjustment for possible confounders. Urine osmolality and htTKV were also associated with eGFR, whereas no association of CC16, NGAL and UMOD with eGFR or htTKV was found.

Conclusion

UACR and urinary KIM-1 are independently associated with kidney size but not with renal function in our study population. Urine osmolality was associated with eGFR and kidney volume following adjustment for multiple confounders. Despite statistical significance, the clinical value of our results is not yet conceivable. Further studies are needed to evaluate the property of the aforementioned biomarkers to assess disease state at early ADPKD stage.     

Assessment of the Effect of Intestinal Permeability Probes (Lactulose And Mannitol) and Other Liquids on Digesta Residence Times in Various Segments of the Gut Determined by Wireless Motility Capsule: A Randomised Controlled Trial

Background

Whilst the use of the mannitol/lactulose test for intestinal permeability has been long established it is not known whether the doses of these sugars modify transit time Similarly it is not known whether substances such as aspirin that are known to increase intestinal permeability to lactulose and mannitol and those such as ascorbic acid which are stated to be beneficial to gastrointestinal health also influence intestinal transit time.

Methods

Gastric and intestinal transit times were determined with a SmartPill following consumption of either a lactulose mannitol solution, a solution containing 600 mg aspirin, a solution containing 500 mg of ascorbic acid or an extract of blackcurrant, and compared by doubly repeated measures ANOVA with those following consumption of the same volume of a control in a cross-over study in six healthy female volunteers. The dominant frequencies of cyclic variations in gastric pressure recorded by the Smartpill were determined by fast Fourier transforms.

Results

The gastric transit times of lactulose mannitol solutions, of aspirin solutions and of blackcurrant juice did not differ from those of the control. The gastric transit times of the ascorbic acid solutions were significantly shorter than those of the other solutions. There were no significant differences between the various solutions either in the total small intestinal or colonic transit times. The intraluminal pHs during the initial quartiles of the small intestinal transit times were lower than those in the succeeding quartiles. This pattern did not vary with the solution that was consumed. The power of the frequencies of cyclic variation in intragastric pressure recorded by the Smartpill declined exponentially with increase in frequency and did not peak at the reported physiological frequencies of gastric contractile activity.

Conclusions

Whilst the segmental residence times were broadly similar to those using other methods, the high degree of variation between subjects generally precluded the identification of all but gross variation between treatments. The lack of any differences between treatments in either total small or large intestinal transit times indicates that the solutions administered in the lactulose mannitol test of permeability had no consistent influence on the temporal pattern of absorption. The negatively exponential profile and lack of any peaks in the frequency spectra of cyclic variation in gastric intraluminal pressure that were consistent with reported physiological frequencies of contractile activity profile suggests that the principal source of this variation is stochastic likely resulting from the effects of external events occasioned by normal daily activities on intra-abdominal pressure.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12615000596505     

Revealing Different Roles of the mTOR-Targets S6K1 and S6K2 in Breast Cancer by Expression Profiling and Structural Analysis

Background

The AKT/mTORC1/S6K pathway is frequently overstimulated in breast cancer, constituting a promising therapeutic target. The benefit from mTOR inhibitors varies, likely as a consequence of tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms. The mTORC1 downstream effectors S6K1, S6K2, and 4EBP1 are amplified and overexpressed in breast cancer, associated with a poor outcome and divergent endocrine treatment benefit. S6K1 and S6K2 share high sequence homology, but evidence of partly distinct biological functions is emerging. The aim of this work was to explore possible different roles and treatment target potentials of S6K1 and S6K2 in breast cancer.

Materials and methods

Whole-genome expression profiles were compared for breast tumours expressing high levels of S6K1, S6K2 or 4EBP1, using public datasets, as well as after in vitro siRNA downregulation of S6K1 and/or S6K2 in ZR751 breast cancer cells. In silico homology modelling of the S6K2 kinase domain was used to evaluate its possible structural divergences to S6K1.

Results

Genome expression profiles were highly different in S6K1 and S6K2 high tumours, whereas S6K2 and 4EBP1 profiles showed significant overlaps, both correlated to genes involved in cell cycle progression, among these the master regulator E2F1. S6K2 and 4EBP1 were inversely associated with IGF1 levels, and their prognostic value was shown to be restricted to tumours positive for IGFR and/or HER2. In vitro, S6K1 and S6K2 silencing resulted in upregulation of genes in the mTORC1 and mTORC2 complexes. Isoform-specific silencing also showed distinct patterns, e.g. S6K2 downregulation lead to upregulation of several cell cycle associated genes. Structural analyses of the S6K2 kinase domain showed unique structure patterns, deviating from those of S6K1, facilitating the development of isoform-specific inhibitors. Our data support emerging proposals of distinct biological features of S6K1 and S6K2, suggesting their importance as separate oncogenes and clinical markers, where specific targeting in different breast cancer subtypes could facilitate further individualised therapies.     

Current Efavirenz (EFV) or Ritonavir-Boosted Lopinavir (LPV/r) Use Correlates with Elevate Markers of Atherosclerosis in HIV-Infected Subjects in Addis Ababa,Ethiopia

Background

HIV patients on antiretroviral therapy have shown elevated incidence of dyslipidemia, lipodystrophy, and cardiovascular disease (CVD). Most studies, however, focus on cohorts from developed countries, with less data available for these co-morbidities in Ethiopia and sub-Saharan Africa.

Methods

Adult HIV-negative (n = 36), treatment naïve (n = 51), efavirenz (EFV)-treated (n = 91), nevirapine (NVP)-treated (n = 95), or ritonavir-boosted lopinavir (LPV/r)-treated (n=44) subjects were recruited from Black Lion Hospital in Addis Ababa, Ethiopia. Aortic pressure, augmentation pressure, and pulse wave velocity (PWV) were measured via applanation tonometry and carotid intima-media thickness (cIMT) and carotid arterial stiffness, and brachial artery flow-mediated dilation (FMD) were measured via non-invasive ultrasound. Body mass index, waist-to-hip circumference ratio (WHR), skinfold thickness, and self-reported fat redistribution were used to quantify lipodystrophy. CD4+ cell count, plasma HIV RNA levels, fasting glucose, total-, HDL-, and LDL-cholesterol, triglycerides, hsCRP, sVCAM-1, sICAM-1, leptin and complete blood count were measured.

Results

PWV and normalized cIMT were elevate and FMD impaired in EFV- and LPV/r-treated subjects compared to NVP-treated subjects; normalized cIMT was also elevated and FMD impaired in the EFV- and LPV/r-treated subjects compared to treatment-naïve subjects. cIMT was not statistically different across groups. Treated subjects exhibited elevated markers of dyslipidemia, inflammation, and lipodystrophy. PWV was associated with age, current EFV and LPV/r used, heart rate, blood pressure, triglycerides, LDL, and hsCRP, FMD with age, HIV duration, WHR, and glucose, and cIMT with age, current EFV use, skinfold thickness, and blood pressure.

Conclusions

Current EFV- or LPV/r-treatment, but not NVP-treatment, correlated with elevated markers of atherosclerosis, which may involve mechanisms distinct from traditional risk factors.     

A Mutation in the Mitochondrial Fission Gene Dnm1l Leads to Cardiomyopathy

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.