Endotoxin tolerance represents a distinctive state of alternative polarization (M2) in human mononuclear cells

Classical (M1) and alternative (M2) polarization of mononuclear cells (MNCs) such as monocyte and macrophages is known to occur in response to challenges within a microenvironment, like the encounter of a pathogen. LPS, also known as endotoxin, is a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in MNCs known as endotoxin tolerance, defined as the reduced capacity of a cell to respond to LPS activation after an initial exposure to this stimulus. Using systems biology approaches in PBMCs, monocytes, and monocyte-derived macrophages involving microarrays and advanced bioinformatic analysis, we determined that gene responses during endotoxin tolerance were similar to those found during M2 polarization, featuring gene and protein expression critical for the development of key M2 MNC functions, including reduced production of proinflammatory mediators, expression of genes involved in phagocytosis, as well as tissue remodeling. Moreover, expression of different metallothionein gene isoforms, known for their role in the control of oxidative stress and in immunomodulation, were also found to be consistently upregulated during endotoxin tolerance. These results demonstrate that after an initial inflammatory stimulus, human MNCs undergo an M2 polarization probably to control hyperinflammation and heal the affected tissue.     

Mast cell accumulation in glioblastoma with a potential role for stem cell factor and chemokine CXCL12

Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf-/- mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma.     

Light-harvesting complex II pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella

 Like higher plants, unicellular green algae of the genus Dunaliella respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light-inducible proteins (Elips) in plants. Earlier studies indicated that Cbr was associated with the light-harvesting complex of photosystem II (LHCII) and suggested it acted as a zeaxanthin-binding protein and fulfilled a photo-protective function (Levy et al. 1993, J. Biol. Chem. 268: 20892–20896). To characterize the protein-pigment subcomplexes containing Cbr in greater detail than attained so far, thylakoid membranes from Dunaliella salina grown in high light or normal light were solubilized with dodecyl maltoside and fractionated by isoelectric-focusing. Analysis of the resolved LHCII subcomplexes indicated preferred associations among the four LHCIIb polypeptides and between them and Cbr: subcomplexes including Cbr contained one or two of the more acidic of the four LHCIIb polypeptides as well as large amounts of lutein and zeaxanthin relative to chlorophyll a/b. After sucrose gradient centrifugation, Cbr free of LHCIIb polypeptides was detected together with released pigments; this Cbr possibly originated in subcomplexes dissociated in the course of the analysis. These results agree with the conclusion that Cbr is part of the network of LHCIIb protein-pigment complexes and suggest that the role played by Cbr involves the organization and/or stabilization of assemblies highly enriched in zeaxanthin and lutein. Such assemblies may function to protect PSII from photodamage due to overexcitation. Received: 6 August 1999 / Accepted: 23 November 1999     

Fermentable carbohydrate alters hypothalamic neuronal activity and protects against the obesogenic environment

Obesity has become a major global health problem. Recently, attention has focused on the benefits of fermentable carbohydrates on modulating metabolism. Here, we take a system approach to investigate the physiological effects of supplementation with oligofructose-enriched inulin (In). We hypothesize that supplementation with this fermentable carbohydrate will not only lead to changes in body weight and composition, but also to modulation in neuronal activation in the hypothalamus. Male C57BL/6 mice were maintained on a normal chow diet (control) or a high fat (HF) diet supplemented with either oligofructose-enriched In or corn starch (Cs) for 9 weeks. Compared to HF+Cs diet, In supplementation led to significant reduction in average daily weight gain (mean ± s.e.m.: 0.19 ± 0.01 g vs. 0.26 ± 0.02 g, P < 0.01), total body adiposity (24.9 ± 1.2% vs. 30.7 ± 1.4%, P < 0.01), and lowered liver fat content (11.7 ± 1.7% vs. 23.8 ± 3.4%, P < 0.01). Significant changes were also observed in fecal bacterial distribution, with increases in both Bifidobacteria and Lactobacillius and a significant increase in short chain fatty acids (SCFA). Using manganese-enhanced MRI (MEMRI), we observed a significant increase in neuronal activation within the arcuate nucleus (ARC) of animals that received In supplementation compared to those fed HF+Cs diet. In conclusion, we have demonstrated for the first time, in the same animal, a wide range of beneficial metabolic effects following supplementation of a HF diet with oligofructose-enriched In, as well as significant changes in hypothalamic neuronal activity.     

Plasma membrane electron transport coupled to Na(+) extrusion in the halotolerant alga Dunaliella

The halotolerant alga Dunaliella adapts to exceptionally high salinity and maintains low [Na(+)](in) at hypersaline solutions, suggesting that it possesses efficient mechanisms for regulating intracellular Na(+). In this work we examined the possibility that Na(+) export in Dunaliella is linked to a plasma membrane electron transport (redox) system. Na(+) extrusion was induced in Dunaliella cells by elevation of intracellular Na(+) with Na(+)-specific ionophores. Elevation of intracellular Na(+) was found to enhance the reduction of an extracellular electron acceptor ferricyanide (FeCN). The quinone analogs NQNO and dicumarol inhibited FeCN reduction and led to accumulation of Na(+) by inhibition of Na(+) extrusion. These inhibitors also diminished the plasma membrane potential in Dunaliella. Anaerobic conditions elevated, whereas FeCN partially decreased intracellular Na(+) content. Cellular NAD(P)H level decreased upon enhancement of plasma membrane electron transport. These results are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to Na(+) extrusion in Dunaliella plasma membrane. We propose that redox-driven Na(+) extrusion and recycling in Dunaliella evolved as means of adaptation to hypersaline environments.     

Expression analysis of buckwheat (Fagopyrum esculentum Moench) metallothionein-like gene (MT3) under different stress and physiological conditions

The buckwheat metallothionein-like (MT3) gene expression was studied throughout seed and leaf development, as well as under the influence of different external stimuli. MT3 mRNAs were detected from the early stage of seed development to the end of maturation, reaching the highest level during the mid-maturation stage. High MT3 mRNA level was noticed for both green and senescent leaves. The influence of raising Cu ion concentrations on MT3 gene expression was studied only in leaves, while the effect of Zn ions was analyzed through seed development as well. It was found that Cu and Zn ions had stimulatory effects on expression in leaves. MT3 expression was significantly enhanced in the early stage of seed development in response to Zn ions, while after this stage, influence of Zn ions was not detected. After H2O2/NaCl treatment, MT3 mRNA level was decreased in green leaves, contrary to senescent leaves where expression levels remained unchanged. H2O2 treatment caused the increase of MT3 mRNA levels in the mid-maturation stage of seed development. NaCl had no effect on expression levels in seeds. According to obtained results, proposed functions in different plant organs regarding oxidative stress and metal homeostasis are discussed.     

Structure of the integrin binding fragment from fibrillin-1 gives new insights into microfibril organization

Human fibrillin-1, the major structural protein of extracellular matrix (ECM) 10-12 nm microfibrils, is dominated by 43 calcium binding epidermal growth factor-like (cbEGF) and 7 transforming growth factor beta binding protein-like (TB) domains. Crystal structures reveal the integrin binding cbEGF22-TB4-cbEGF23 fragment of human fibrillin-1 to be a Ca(2+)-rigidified tetragonal pyramid. We suggest that other cbEGF-TB pairs within the fibrillins may adopt a similar orientation to cbEGF22-TB4. In addition, we have located a flexible RGD integrin binding loop within TB4. Modeling, cell attachment and spreading assays, immunocytochemistry, and surface plasmon resonance indicate that cbEGF22 bound to TB4 is a requirement for integrin activation and provide insight into the molecular basis of the fibrillin-1 interaction with alphaVbeta3. In light of our data, we propose a novel model for the assembly of the fibrillin microfibril and a mechanism to explain its extensibility.     

The fate of the arachnoid villi in humans

Villi arachnoidales undergoes in the course of life changes in relation to the skull bones and sinuses. Our aim was to determine the relations of the villi arachnoidales to the skull bone and/or sinuses from the neonatal period to adults. The investigations were performed on collection of 50 disarticulated macerated skull bones from the new-born to 30 years of age and on 20 skulls from individuals in the life period from 30 to 80 years of age. Villi arachnoidales produced imprints on the skull bones in the shape of holes and/or furrows corresponding to different shape of the villi arachnoidales. These imprints appeared very early in the period when the bony sprouts of the large skull bones received a thin covering of compact bone, the future lamina vitrea. At that time villi arachnoidales had no connection with the dural sinuses but with the diploe and with the diploic veins. By agglomeration of the villi in larger and large formations, granula meningea, Pacchionian granulations, the contact to sinuses was realized by means of short channels. The structural changes of villi arachnoidales may produce thrombophlebitis and hydrocephalus externus, especially in children. The fate and the relations of the villi arachnoidales are therefore of great importance for neurologist, neurosurgeon and otorhinolaryngologist.     

Preliminary investigation: the impact of the NCAA Wrestling Weight Certification Program on weight cutting

Historically, wrestling is a sport dependent on weight. Three tragic deaths in late 1997 prompted the National Collegiate Athletic Association (NCAA) to make a Wrestling Weight Certification Program (WWCP) mandatory to foster a safe competitive environment. One institution examined the impact of this program on weight cutting. Thirty-two NCAA Division I wrestlers completed the WWCP in the 1998-1999 season and 29 in 1999-2000. Eighteen (56%) of 32 wrestlers in 1998-1999 weighed in 10 or more pounds above the previous year's competition weight. Whereas, 28% weighed in 20 or more pounds above the previous year's competition weight. Weekly weight loss for the wrestlers in 1998-1999 revealed a substantial loss during the first week, possibly demonstrating the use of time-tested techniques for weight loss. However, in 1999-2000, the first week weight loss was less pronounced, with 65.8% of the weight being lost during the second half of the WWCP. Therefore, these wrestlers may be breaking the sport historic cycle of weight fluctuations through the WWCP.