Fine specificity and antigen receptor expression among influenza virus-specific cytolytic T lymphocyte clones

Influenza virus stimulates a vigorous cytolytic T lymphocyte (CTL) response in the mouse that is directed to several virion polypeptides. This report examines the fine specificity of a panel of murine influenza-specific CTL clones restricted by MHC class I products of the H-2d haplotype. Ten of 22 A/JAPAN/305/57-specific CTL clones analyzed were directed to the A/JAPAN/305/57 hemagglutinin protein as detected by using target cells infected with a recombinant vaccinia virus containing hemagglutinin gene. Based on their fine specificity of hemagglutinin recognition, these clones defined four functional epitopes on the hemagglutinin. The remaining 12 cytolytic clones exhibited cross-reactivity for type A influenza viruses of the major human subtypes, and approximately 60% of these clones were directed to the nucleocapsid protein. KJ16-133 monoclonal antibody analysis of the utilization of the T cell receptor V beta 8 gene segment subfamily revealed that members of this V beta gene subfamily are expressed by both hemagglutinin- and nucleocapsid-specific MHC class I-restricted CTL (and by influenza-specific MHC class II-restricted T lymphocytes as well). These results suggest that CTL detect several distinct antigenic sites on the hemagglutinin. In addition, these results reveal no direct correlation between viral antigenic specificity and V beta gene expression by these virus-specific CLT clones.     

Towards global patterns in the diversity and community structure of ectomycorrhizal fungi

Global species richness patterns of soil micro-organisms remain poorly understood compared to macro-organisms. We use a global analysis to disentangle the global determinants of diversity and community composition for ectomycorrhizal (EcM) fungi-microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro-organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next-generation sequencing techniques and relevant soil metadata.     

Patterns of benthic mega-invertebrate habitat associations in the Pacific Northwest continental shelf waters: a reassessment

As human impacts and demands for ocean space increase (fisheries, aquaculture, marine reserves, renewable energy), identification of marine habitats hosting sensitive biological assemblages has become a priority. Epifaunal invertebrates, especially the structure-forming species, are an increasing conservation concern as many traditional (bottom-contact fishing) and novel (marine renewable energy) ocean uses have the potential to displace or otherwise impact these slow-growing organisms. The differences in mega-invertebrate species assemblages between high-relief rocks and low-relief sediments are well documented and likely hold for most marine environments. In anticipation of potential development of marine renewable energy faculties off Oregon and Washington (USA), a survey of the benthic invertebrate assemblages and habitats was conducted on three rocky reefs on the continental shelf of the Pacific Northwest, using video footage collected by remotely operated vehicle, to more finely characterize these assemblage–habitat associations. Benthic assemblages appeared to first group by depth (50–80 vs. 100–120 m), then by relief (consolidated rocks vs. unconsolidated rocks and soft sediments). Consolidated rocks were characterized at each site by a combination of various sponges, gorgonians, sea anemones and echinoderms; unconsolidated rocks were characterized at Grays Bank by sea anemones and burrowing brittle stars, and at Bandon-Arago by sponges and echinoderms; soft sediments were characterized at Grays Bank and Siltcoos Reef by sea whips and burrowing brittle stars, as well as pink shrimps and sea stars at Siltcoos Reef, and at Bandon-Arago by sponges, gorgonians and echinoderms. The results of this study will help classify and map the seafloor in a way that represents benthic habitats reflective of biological species assemblage distributions, rather than solely geological features, and support conservation and management planning.     

Excess circulating alternatively activated myeloid (M2) cells accelerate ALS progression while inhibiting experimental autoimmune encephalomyelitis

Background

Circulating immune cells including autoreactive T cells and monocytes have been documented as key players in maintaining, protecting and repairing the central nervous system (CNS) in health and disease. Here, we hypothesized that neurodegenerative diseases might be associated, similarly to tumors, with increased levels of circulating peripheral myeloid derived suppressor cells (MDSCs), representing a subset of suppressor cells that often expand under pathological conditions and inhibit possible recruitment of helper T cells needed for fighting off the disease.

Methods and Findings

We tested this working hypothesis in amyotrophic lateral sclerosis (ALS) and its mouse model, which are characterized by a rapid progression once clinical symptoms are evident. Adaptive transfer of alternatively activated myeloid (M2) cells, which homed to the spleen and exhibited immune suppressive activity in G93A mutant superoxide dismutase-1 (mSOD1) mice at a stage before emergence of disease symptoms, resulted in earlier appearance of disease symptoms and shorter life expectancy. The same protocol mitigated the inflammation-induced disease model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), which requires circulating T cells for disease induction. Analysis of whole peripheral blood samples obtained from 28 patients suffering from sporadic ALS (sALS), revealed a two-fold increase in the percentage of circulating MDSCs (LIN^−/LowHLA-DR⁻CD33⁺) compared to controls.

Conclusions

Taken together, these results emphasize the distinct requirements for fighting the inflammatory neurodegenerative disease, multiple sclerosis, and the neurodegenerative disease, ALS, though both share a local inflammatory component. Moreover, the increased levels of circulating MDSCs in ALS patients indicates the operation of systemic mechanisms that might lead to an impairment of T cell reactivity needed to overcome the disease conditions within the CNS. This high level of suppressive immune cells might represent a risk factor and a novel target for therapeutic intervention in ALS at least at the early stage.     

Direct observation of membrane retrieval in chromaffin cells by capacitance measurements

This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)-membrane components from the plasma membrane. For this purpose, 'on-cell' capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo- and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin-coated vesicles, at approximately the same ratio. These results were confirmed by rapid-freeze immuno-electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.     

From proteomic multimarker profiling to interesting proteins: thymosin-β(4) and kininogen-1 as new potential biomarkers for inflammatory hepatic lesions

Despite tremendous efforts in disclosing the pathophysiological and epidemiological factors associated with liver fibrogenesis, non-invasive diagnostic measures to estimate the clinical outcome and progression of liver fibrogenesis are presently limited. Therefore, there is a mandatory need for methodologies allowing the reasonable and reliable assessment of the severity and/or progression of hepatic fibrogenesis. We here performed proteomic serum profiling by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in 179 samples of patients chronically infected with hepatitis C virus and 195 control sera. Multidimensional analysis of spectra allowed the definition of algorithms capable to distinguish class-specific protein expression profiles in serum samples. Overall about 100 peaks could be detected per single spectrum. Different algorithms including protein peaks in the range of 2000 and 10,000 Da were generated after pre-fractionation on a weak cation exchange surface. A specificity of 93% with a sensitivity of 86% as mean of the test set results was found, respectively. The nature of three of these protein peaks that belonged to kininogen-1 and thymosin-β(4) was further analysed by tandem mass spectrometry (MS)/MS. We further found that kininogen-1 mRNA was significantly down-regulated in cirrhotic livers. We have identified kininogen-1 and thymosin-β(4) as potential new biomarkers for human chronic hepatitis C and conclude that serum profiling is a reliable technique to identify hepatitis-associated expression patterns. Based on the high throughput capability, the identified differential protein panel may serve as a diagnostic marker and warrants further validation in larger cohorts.     

Functional claudication distance: a reliable and valid measurement to assess functional limitation in patients with intermittent claudication

Background

Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS) function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells.

Methods

Human endothelial cells were isolated from umbilical veins and stimulated with TNFα (10 ng/ml) for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin were assessed by immunoblotting.

Results

The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFα was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFα-induced activation of NF-κB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFα.

Conclusion

The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.     

A preliminary study in osteoinduction by a nano-crystalline hydroxyapatite in the mini pig

To test the probable osteoinductive properties of NanoBone, a new highly non-sintered porous nano-crystalline hydroxylapatite bone substitute embedded into a silica gel matrix, granules were implanted subcutaneously and intramuscularly into the back region of 18 mini pigs. After periods of 5 and 10 weeks as well as 4 and 8 months, implantation sites were investigated using histological and histomorphometric procedures. Signs of early osteogenesis could already be detected after 5 weeks. The later periods were characterized by increasing membranous osteogenesis in and around the granules leading to the formation of bone-like structures showing periosteal and tendon-like structures with bone marrow and focal chondrogenesis. Bone formation was better in the subcutaneous than in the intramuscular implantation sites. This ectopic osteogenesis is discussed with regard to the nanoporosity and microporosity of the material, physico-chemical interactions at its surface, the differentiation of osteoblasts, the role of angiogenesis and the probable involvement of growth factors. The results of this preliminary study indicate that this biomaterial has osteoinductive potential and induces the formation of bone structures, mainly in subcutaneous adipose tissue in the pig.     

The mycorrhizal status of Pseudotulostoma volvata (Elaphomycetaceae, Eurotiales, Ascomycota)

Pseudotulostoma volvata (O. K. Mill. and T. W. Henkel) is a morphologically unusual member of the otherwise hypogeous Elaphomycetaceae due to its epigeous habit and exposed gleba borne on an elevated stalk at maturity. Field observations in Guyana indicated that P. volvata was restricted to rain forests dominated by ectomycorrhizal (EM) Dicymbe corymbosa (Caesalpiniaceae), suggesting an EM nutritional mode for the fungus. In this paper, we confirm the EM status of P. volvata with a combination of morphological, molecular, and mycosociological data. The EM status for P. volvata corroborates its placement in the ectotrophic Elaphomycetaceae.     

Guyanagaster, a new wood-decaying sequestrate fungal genus related to Armillaria (Physalacriaceae, Agaricales, Basidiomycota)

? Premise of the study: Sequestrate basidiomycete fungi (e.g. "gasteromycetes") have foregone ballistospory and evolved alternative, often elaborate mechanisms of basidiospore dispersal with highly altered basidioma morphology. Sequestrate fungi have independently evolved in numerous Agaricomycete lineages, confounding taxonomic arrangements of these fungi for decades. Understanding the multiple origins and taxonomic affinities of sequestrate fungi provides insight into the evolutionary forces that can drastically alter basidioma morphology. In the neotropical rainforests of the Guiana Shield, we encountered a remarkable sequestrate fungus fruiting directly on decaying hardwood roots. The fungus' singular combination of traits include a wood-decaying habit; black, verrucose peridium; reduced stipe; and gelatinized basidiospore mass. ? Methods: Guyanagaster necrorhiza gen. et sp. nov. is described. Macro- and micromorphological characters were assessed and compared to most similar taxa. To determine the phylogenetic affinities of the fungus, DNA sequence data were obtained for the 18S, ITS, and 28S rDNA, RBP2, and EF1α regions and subjected to single- and multi-gene analyses. DNA sequences from fungal vegetative organs growing on decaying woody roots confirmed the wood-inhabiting lifestyle of Guyanagaster. ? Key results: Guyanagaster is morphologically unique among sequestrate fungi worldwide. Phylogenetic evidence places Guyanagaster in close relation to the wood-decaying mushroom genus Armillaria in the Physalacriaceae (Agaricales, Agaricomycetes, Basidiomycota). ? Conclusions: Guyanagaster represents an independently evolved sequestrate form within the Physalacriaceae. Although molecular data confirm that Guyanagaster is closely related to Armillaria, the unusual features of this fungus suggest a case of radically divergent morphological evolution.