Enhancement of Tight Junctional Barrier Function by Micronutrients: Compound-Specific Effects on Permeability and Claudin Composition

Amid an increasing number of reports in the literature concerning epithelial barrier enhancement by various nutrient compounds, there has never been a study performing side-by-side comparisons of these agents in a single epithelial model. We compare five nutrient compounds (previously reported in various epithelial models to enhance barrier function) regarding their ability to increase transepithelial electrical resistance (R_t) and decrease transepithelial mannitol permeability (J_m) across LLC-PK₁ renal epithelial cell layers. The effects of these nutrients on the abundance of various tight junctional proteins are also compared. In the overall group of nutrients tested - zinc, indole, quercetin, butyrate and nicotine - only nicotine failed to improve barrier function by either parameter. Nicotine also was without effect on tight junctional proteins. Quercetin simultaneously increased R_t and decreased J_m. Zinc, butyrate and indole only exhibited statistically significant enhancement of R_t. Each of these four effective nutrient compounds had unique patterns of effects on the panel of tight junctional proteins studied. No two compounds produced the same pattern of effects. This unique pattern of effects on tight junctional complex composition by each compound establishes the chance for additive or even synergistic improvement of barrier function by combinations of compounds. A synergistic effect of the combination of quercetin and zinc on R_t is shown.     

Effect of temperature and light on seed germination of Erysimum naxense and Erysimum krendlii

Seed germination of two local Greek endemics was studied (Erysimum naxense, Erysimum krendlii). Seed viability was determined by using the tetrazolium method and germination was studied in synchronized cycles of five and four alternating temperatures [10/5 (for E. naxense only) and 15/10, 20/15, 25/20, and 30/25°C for both species, in cycles of 16 h day/8 h night], and in five light regimes (red, blue, green, white, and dark). Germination of E. naxense and E. krendlii seeds was determined daily for six and five weeks, respectively, with the data analyzed as viability adjusted accumulative seed germination at the end of each week. E. naxense’s seed viability was higher (90%) than that of E. krendlii (64%); seed germination (%) of both increased at low alternating temperatures (10/5°C, 15/10°C, 20/15°C). Germination of E. naxense seeds at low temperatures was light-independent, whereas at high temperatures it was increased with red light. Germination of E. krendlii seeds was inconsistently affected by light at the temperatures studied. Percentages of seed germination of both species were higher in experimental conditions similar to the ones of their natural habitats during autumn and/or spring (facilitated with Geographic Information Systems). These conclusions provide guidelines for species-specific propagation protocols and ex situ conservation.     

Ixabepilone Administered Weekly or Every Three Weeks in HER2-Negative Metastatic Breast Cancer Patients; A Randomized Non-Comparative Phase II Trial

To explore the activity and safety of two schedules of ixabepilone, as first line chemotherapy, in patients with metastatic breast cancer previously treated with adjuvant chemotherapy, a randomized non-comparative phase II study was conducted. From November 2008 until December 2010, 64 patients were treated with either ixabepilone 40 mg/m² every 3 weeks (Group A, 32 patients) or ixabepilone 20 mg/m² on days 1, 8 and 15 every 4 weeks (Group B, 32 patients). Overall response rate (the primary end point) was 47% in Group A and 50% in Group B. The most frequent severe adverse events were neutropenia (32% vs. 23%), metabolic disturbances (29% vs. 27%) and sensory neuropathy (12% vs. 27%). Two patients in Group A and 3 in Group B developed febrile neutropenia. After a median follow-up of 22.7 months, median progression-free survival (PFS) was 9 months in Group A and 12 months in Group B. Median survival was 26 months in Group A, whereas it was not reached in Group B. Multiple genetic and molecular markers were examined in tumor and peripheral blood DNA, but none of them was associated with ORR or drug toxicity. Favorable prognostic markers included: the T-variants of ABCB1 SNPs c.2677G/A/T, c.1236C/T and c.3435C/T, as well as high MAPT mRNA and Tau protein expression, which were all associated with the ER/PgR-positive phenotype; absence of TopoIIa; and, an interaction between low TUBB3 mRNA expression and Group B. Upon multivariate analysis, tumor ER-positivity was a favorable (p = 0.0092) and TopoIIa an unfavorable (p = 0.002) prognostic factor for PFS; PgR-positivity was favorable (p = 0.028) for survival. In conclusion, ixabepilone had a manageable safety profile in both the 3-weekly and weekly schedules. A number of markers identified in the present trial appear to deserve further evaluation for their prognostic and/or predictive value in larger multi-arm studies.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT 00790894","term_id":"NCT00790894"}}NCT 00790894     

Importance of Ethnicity,CYP2B6 and ABCB1 Genotype for Efavirenz Pharmacokinetics and Treatment Outcomes: A Parallel-Group Prospective Cohort Study in Two Sub-Saharan Africa Populations

Objectives

We evaluated the importance of ethnicity and pharmacogenetic variations in determining efavirenz pharmacokinetics, auto-induction and immunological outcomes in two African populations.

Methods

ART naïve HIV patients from Ethiopia (n = 285) and Tanzania (n = 209) were prospectively enrolled in parallel to start efavirenz based HAART. CD4+ cell counts were determined at baseline, 12, 24 and 48 weeks. Plasma and intracellular efavirenz and 8-hydroxyefvairenz concentrations were determined at week 4 and 16. Genotyping for common functional CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 variant alleles were done.

Result

Patient country, CYP2B6*6 and ABCB1 c.4036A>G (rs3842A>G) genotype were significant predictors of plasma and intracellular efavirenz concentration. CYP2B6*6 and ABCB1 c.4036A>G (rs3842) genotype were significantly associated with higher plasma efavirenz concentration and their allele frequencies were significantly higher in Tanzanians than Ethiopians. Tanzanians displayed significantly higher efavirenz plasma concentration at week 4 (p<0.0002) and week 16 (p = 0.006) compared to Ethiopians. Efavirenz plasma concentrations remained significantly higher in Tanzanians even after controlling for the effect of CYP2B6*6 and ABCB1 c.4036A>G genotype. Within country analyses indicated a significant decrease in the mean plasma efavirenz concentration by week 16 compared to week 4 in Tanzanians (p = 0.006), whereas no significant differences in plasma concentration over time was observed in Ethiopians (p = 0.84). Intracellular efavirenz concentration and patient country were significant predictors of CD4 gain during HAART.

Conclusion

We report substantial differences in efavirenz pharmacokinetics, extent of auto-induction and immunologic recovery between Ethiopian and Tanzanian HIV patients, partly but not solely, due to pharmacogenetic variations. The observed inter-ethnic variations in efavirenz plasma exposure may possibly result in varying clinical treatment outcome or adverse event profiles between populations.     

Atherosclerotic cardiovascular disease and dermatomyositis: an analysis of the Nationwide Inpatient Sample survey

Introduction

Increased rates of cardiovascular disease are implicated in several rheumatologic diseases. Our aim was to characterize dermatomyositis hospitalizations and evaluate cardiovascular-associated mortality in this patient population.

Methods

We examined the frequency and mortality rates of several atherosclerotic cardiovascular diagnoses and procedures among hospitalized adult patients with dermatomyositis using data from the US Nationwide Inpatient Sample (NIS) from 1993 to 2007. We compared the odds of death among hospitalized dermatomyositis patients with each cardiovascular diagnosis or procedure to those without, as well as to controls with cardiovascular diagnoses, using logistic regression.

Results

A total of 50,322 hospitalizations of dermatomyositis patients occurred between 1993 and 2007 (mean age 58 years, and 73% female). Of all dermatomyositis hospitalizations, 20% were associated with a concurrent atherosclerotic cardiovascular diagnosis or procedure. The overall in-hospital mortality was 5.7%. Dermatomyositis patients with any associated atherosclerotic cardiovascular diagnosis or procedure were twice as likely to die during the inpatient stay compared to dermatomyositis patients who did not have atherosclerotic cardiovascular disease (OR = 2.0 95% CI 1.7-2.5, p < 0.0001). The odds ratio for death in patients with both dermatomyositis and cardiovascular disease compared to controls with cardiovascular disease alone was 1.98 (95% CI 1.57-2.48) in multivariate adjusted models.

Conclusions

Approximately one fifth of dermatomyositis hospitalizations in the US were associated with an atherosclerotic cardiovascular diagnosis or procedure. These patients have double the risk of in-hospital death in comparison with controls and dermatomyositis patients without a cardiovascular diagnosis, making identification of these groups important for both prognostic purposes and clinical care.     

Characterization of nitrogen-fixing bacteria isolated from field-grown barley,oat, and wheat

Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.     

Cell Survival during Complete Nutrient Deprivation Depends on Lipid Droplet-fueled β-Oxidation of Fatty Acids

Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A₂ (cPLA₂α) or down-regulation of ceramide kinase. Nutrient deprivation also induced β-oxidation of fatty acids that was sensitive to cPLA₂α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by β-oxidation of fatty acids that requires biogenesis and mobilization of LD.     

Harmful and parasitic unicellular eukaryotes persist in a shallow lake under reconstruction (L. Karla, Greece)

The reconstructed Lake Karla, Greece, has been undergoing its water-filling period since November 2009. In this paper, we aimed at investigating whether the unicellular eukaryotes, including the toxic/parasitic ones, that have been found during mass fish kills in the lake (March–April 2010), persist during the first warm period of the lake (May, August, November 2010). Given that microscopic characterization of some of these eukaryotes is not adequate for their identification, we analysed the 18S rRNA gene diversity of plankton samples. All the found phylotypes belonged to the phyla of Mesomycetazoa, Chlorophyta, Fungi, Alveolata, Cercozoa, Cryptophyta and Stramenopiles. Some members of these groups seem to persist in Lake Karla as they have been found in early spring as well. These microscopic eukaryotes are either ichthyotoxic/parasitic (e.g. Pfiesteria sp./Pseudopfiesteria shumwayae, some Fungi, Mesomycetazoa, Lagenidium sp., Cercozoa) or indicative of hyper-eutrophic conditions (e.g. Oocystis sp., Scenedesmus spp.) and were rather abundant during the first spring–autumn period of the lake’s refilling process. These complex microscopic communities are expected to shape highly dynamic and variable food webs with the risk of repeated fish kills.     

Chroman-like cyclic prenylflavonoids promote neuronal differentiation and neurite outgrowth and are neuroprotective

Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly “neuroactive” compounds, which we termed “enhancement of neuronal differentiation factors” (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments.