Butyrate inhibits and Escherichia coli-derived mitogen(s) stimulate DNA synthesis in human hepatocytes in vitro

Bacterial constituents and products of the bacterial metabolism pass from the gut lumen to the portal vein and may influence the homeostasis of the liver. Our aim is to examine whether DNA synthesis of human hepatocyte cell lines is affected by constituents of Escherichia coli species as well as by intracolonic products of bacterial fermentation that reach the liver via the portal vein. Supernatant solutions and bacterial cell fractions (containing either whole dead bacteria, cell walls, cytosol or non-soluble intracellular components) of E. coli K12 and of E. coli species from rat fecal flora were separated by multi-step centrifugation, French press, and microfiltration. The supernatant solution and the cell fractions were incubated with a human hepatoma cell line (Hep-G2) and with a cell line derived from non-malignant human liver cells (Chang cells) for 24 h. The cells were labeled with tritiated thymidine before processing to autoradiography. DNA synthesis was estimated by the labeling index (LI%). DNA synthesis was also estimated following incubation of Hep-G2 cells with short chain fatty acids (acetic, propionic, butyric and succinic acid), acetaldehyde, and ammonium chloride. Epidermal growth factor and a water extract of Helicobacter pylori were used as references. The fractions of E. coli from rat fecal flora containing cytosol and non-soluble intracellular components significantly increased the labeling index in both Hep-G2 and Chang cells (p < 0.05). In addition, the supernatant solution significantly increased the LI in Chang cells (p < 0.05). Epidermal growth factor increased the LI of Hep-G2 cells dose-dependently (p < 0.05). Butyric acid reduced DNA synthesis at 10(-4) M (p < 0.05). The highest doses of acetaldehyde were cytotoxic and reduced the LI. Escherichia coli species contain mitogenic factors to human hepatocytes. The mitogen(s) are present in the supernatant solution, in the cytosol and in non-soluble intracellular components. Butyrate, which is a product of bacterial fermentation of colonic substrates inhibit DNA synthesis in the hepatocyte cell lines. Our findings suggest that soluble mitogen(s) that diffuse from the microorganism to the outer environment, intracellular bacterial constituents, and products of the bacterial metabolism that reach the liver via the portal vein may influence the cell kinetic steady-state of hepatic cells.     

A simple approximation for larval retention around reefs

Estimating larval retention at individual reefs by local scale three-dimensional flows is a significant problem for understanding, and predicting, larval dispersal. Determining larval dispersal commonly involves the use of computationally demanding and expensively calibrated/validated hydrodynamic models that resolve reef wake eddies. This study models variation in larval retention times for a range of reef shapes and circulation regimes, using a reef-scale three-dimensional hydrodynamic model. It also explores how well larval retention time can be estimated based on the “Island Wake Parameter”, a measure of the degree of flow turbulence in the wake of reefs that is a simple function of flow speed, reef dimension, and vertical diffusion. The mean residence times found in the present study (0.48–5.64 days) indicate substantial potential for self-recruitment of species whose larvae are passive, or weak swimmers, for the first several days after release. Results also reveal strong and significant relationships between the Island Wake Parameter and mean residence time, explaining 81–92% of the variability in retention among reefs across a range of unidirectional flow speeds and tidal regimes. These findings suggest that good estimates of larval retention may be obtained from relatively coarse-scale characteristics of the flow, and basic features of reef geomorphology. Such approximations may be a valuable tool for modeling connectivity and meta-population dynamics over large spatial scales, where explicitly characterizing fine-scale flows around reef requires a prohibitive amount of computation and extensive model calibration.     

Ground beetle (Coleoptera, Carabidae) assemblages inhabiting Scots pine stands of Puszcza Piska Forest: six-year responses to a tornado impact

Ground beetle assemblages were studied during 2003-08 in the Pisz Forest by comparing stands disturbed by a tornado to undisturbed control stands. The following exploratory questions were put forward. (1) How do the carabid assemblages change during six years following the tornado impact? (2) Does the carabid assemblage recovery begin during the six first post-tornado years? To assess the state of carabid assemblages we used two indices: the MIB (Mean Individual Biomass) and the SPC (Sum of Progressive Characteristics). Carabid assemblages in the disturbed and in the control stands, as expressed by these two indices, were compared using the length of a regression distance (sample distance in a MIB:SPC coordinate system). A cluster analysis revealed that the assemblages of the disturbed and the control stands were different. The tornado-impacted stands produced lower carabid catch rates, but species richness was significantly higher there than in the control stands. They hosted lower proportions of individuals of European species, of large zoophages, and of forest and brachypterous species, than the control stands. The observed reduction in SPC and MIB, and an increase in the regression distances may indicate that the carabid assemblages had not started to recover from the tornado-caused disturbance. Carabid assemblages apparently responded to the tornado in two steps. Firstly, the first three years were characterized by moderate decreases of index values. Secondly, from the fourth to the sixth year after the tornado, many observed changes became magnified. We did not observe clear signals of the recovery of forest carabid assemblages during the six follow-up years.     

Effects of temperature on the hill reaction and photophosphorylation in isolated cactus chloroplasts

Chloroplasts isolated from Opuntia polyacantha Haw. (Cactaceae) are capable of noncyclic electron transport and ATP synthesis. Hill reaction rates, measured by O₂ evolution or by ferricyanide reduction, increase with increasing temperature to approximately 40 C. The temperature optimum of NADP reduction is 42 C while the optimum for noncyclic photophosphorylation is 35 C. NADP-linked phosphorylation exhibits a higher coupling ratio (P/e₂) than ferricyanide-linked photophosphorylation. The temperature optima for photochemical energy production correlate with photosynthetic properties of Crassulacean acid metabolism (CAM) plants and are discussed in relation to the operation of CAM at high tissue temperature.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

NETosis occurs independently of neutrophil serine proteases

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.     

The status of the Lutzomyia longipalpis species complex and possible implications for Leishmania transmission

The sand fly Lutzomyia longipalpis sensu latu has been identified as the principal vector of American visceral leishmaniasis, a potentially fatal disease that primarily affects children in several countries of South and Central America. Over the past several years increases have occurred both in the number of reported cases and the population at risk: approximately 1.6 million people reside in highly endemic areas with 16,000 cases reported annually. Several studies have attempted to relate the epidemiology of this disease to variability in Lu. longipalpis that is now recognized to be a complex of at least three sibling species. Morphological variation in this species was first noted by Mangabeira (1969). Since then physiological and biochemical differences have been reported by several investigators. Recent reports in Costa Rica of the presence of Lu. longipalpis in a focus of cutaneous leishmaniasis caused by Leishmania chagasi may be an additional indication of variability in this species. While existing evidence indicates that the morphospecies Lu. longipalpis may represent a complex of sibling species, genetic, epidemiological and ecological distinctions have not been fully resolved. Thus, delimitation of systematic boundaries within the complex and corresponding to geographic distributions and roles in transmission remain unresolved. The purpose of this review is to summarize from the literature observations of polymorphism in this morphospecies and consider what significance this reported variability may have to the epidemiology of visceral leishmaniasis.     

Redox regulation of RyR-mediated Ca2+ release in muscle and neurons

Changes in the redox state of the intracellular ryanodine receptor/Ca2+ release channels of skeletal and cardiac muscle or brain cortex neurons affect their activity. In particular, agents that oxidize or alkylate free SH residues of the channel protein strongly enhance Ca(2+)-induced Ca2+ release, whereas reducing agents have the opposite effects. We will discuss here how modifications of highly reactive cysteine residues by endogenous redox agents or cellular redox state influence RyR channel activation by Ca2+ and ATP or inhibition by Mg2+. Possible physiological and pathological implications of these results on cellular Ca2+ signaling will be addressed as well.