Individual LPS responsiveness depends on the variation of toll-like receptor (TLR) expression level

An individual's immune response is critical for host protection from many different pathogens, and the responsiveness can be assessed by the amount of cytokine production upon stimulating bacterial components such as lipopolysaccharide (LPS). The difference between individuals in their peripheral blood mononuclear cells (PBMC) responsiveness to LPS, a Gram-negative endotoxin, was investigated from 27 healthy individuals. We observed a large variation in IFNgamma production among different individuals. The PBMC of the consistently three highest and three lowest IFNgamma producers were investigated. Since previous studies described that a single point mutation in the coding region of TLR2 and TLR4 is linked to the individual responsiveness to pathogenic bacterial infections, we first examined the known point mutations in the coding region of TLR2Pro681His, TLR4Pro714His located in the cytoplasmic regions of the Toll-like domain as well as TLR4Asp299Gly located in the extracellular region. None of these mutations were associated with an individual's responsiveness to LPS, despite the presence of TLR4Asp299Gly mutation. Further investigation revealed that the variation of PBMC responsiveness to LPS among healthy individuals was due to constitutive expression levels of TLR4 and TLR2. This result is consistent with an aging-related low expression of Toll-like receptors in the mouse model of LPS responsiveness. The present study therefore suggests that the constitutive expression levels of TLR2 and TLR4 may contribute to the individual response to LPS.     

Vertical distribution of picoeukaryotic diversity in the Sargasso Sea

Eukaryotic molecular diversity within the picoplanktonic size-fraction has primarily been studied in marine surface waters. Here, the vertical distribution of picoeukaryotic diversity was investigated in the Sargasso Sea from euphotic to abyssal waters, using size-fractionated samples (< 2 microm). 18S rRNA gene clone libraries were used to generate sequences from euphotic zone samples (deep chlorophyll maximum to the surface); the permanent thermocline (500 m); and the pelagic deep-sea (3000 m). Euphotic zone and deep-sea data contrasted strongly, the former displaying greater diversity at the first-rank taxon level, based on 232 nearly full-length sequences. Deep-sea sequences belonged almost exclusively to the Alveolata and Radiolaria, while surface samples also contained known and putative photosynthetic groups, such as unique Chlorarachniophyta and Chrysophyceae sequences. Phylogenetic analyses placed most Alveolata and Stramenopile sequences within previously reported 'environmental' clades, i.e. clades within the Novel Alveolate groups I and II (NAI and NAII), or the novel Marine Stramenopiles (MAST). However, some deep-sea NAII formed distinct, bootstrap supported clades. Stramenopiles were recovered from the euphotic zone only, although many MAST are reportedly heterotrophic, making the observed distribution a point for further investigation. An unexpectedly high proportion of radiolarian sequences were recovered. From these, five environmental radiolarian clades, RAD-I to RAD-V, were identified. RAD-IV and RAD-V were composed of Taxopodida-like sequences, with the former solely containing Sargasso Sea sequences, although from all depth zones sampled. Our findings highlight the vast diversity of these protists, most of which remain uncultured and of unknown ecological function.     

Development of a bead-based aptamer/antibody detection system for C-reactive protein

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10 mg/L in diluted serum with acceptable recoveries (extrapolated values of 70–130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer–target systems could increase the number of analytes measurable using xMAP-type assays.     

Peripheral blood neutrophil activation patterns are associated with pulmonary inflammatory responses to lipopolysaccharide in humans

Increased nuclear accumulation of NF-kappaB in LPS-stimulated peripheral blood neutrophils has been shown to be associated with more severe clinical course in patients with infection associated acute lung injury. Such observations suggest that differences in neutrophil response may contribute to the pulmonary inflammation induced by bacterial infection. To examine this question, we sequentially measured LPS-induced DNA binding of NF-kappaB in neutrophils collected from healthy humans on at least three occasions, each separated by at least 2 wk, and then determined pulmonary inflammatory responses after instillation of LPS into the lungs. Consistent patterns of peripheral blood neutrophil responses, as determined by LPS-induced NF-kappaB DNA binding, were present in volunteers, with a >80-fold difference between individuals in the mean area under the curve for NF-kappaB activation. The number of neutrophils recovered from bronchoalveolar lavage after exposure to pulmonary LPS was significantly correlated with NF-kappaB activation in peripheral blood neutrophils obtained over the pre-LPS exposure period (r = 0.65, p = 0.009). DNA binding of NF-kappaB in pulmonary neutrophils also was associated with the mean NF-kappaB area under the curve for LPS-stimulated peripheral blood neutrophils (r = 0.63, p = 0.01). Bronchoalveolar lavage levels of IL-6 and TNFRII were significantly correlated with peripheral blood neutrophil activation patterns (r = 0.75, p = 0.001 for IL-6; and r = 0.48, p = 0.049 for TNFRII. These results demonstrate that stable patterns in the response of peripheral blood neutrophils to LPS exist in the human population and correlate with inflammatory response following direct exposure to LPS in the lung.     

Serum Mercury Level and Multiple Sclerosis

Exposure to heavy metals has been associated to a higher incidence of multiple sclerosis. In this work, we present a possible relationship between serum mercury levels and development of multiple sclerosis in Isfahan, the third largest city in Iran. Seventy-four patients affected by multiple sclerosis were retrieved from multiple sclerosis (MS) clinic in Isfahan, Iran. By matching sex and age, 74 healthy volunteers were chosen as control group. Blood samples were collected and serum mercury content was determined. Serum mercury level in MS patients was significantly higher than controls (9.6 ± 10.17 vs. 5.7 ± 8.6, P = 0.037). Concerning all MS patients, serum mercury value was significantly higher than the mercury concentration founded in control subjects {odd ratio: 2.39 (CI, 1.96–2.94), P = 0.00}. Serum mercury level is higher in MS patients with odd ratio equal to 2.39 compared with healthy individuals. It may reveal that high mercury levels in serum might help MS development in susceptible individuals. More studies with larger sample size are needed to confirm this hypothesis.     

Expression of Androgen Receptor and Cancer Stem Cell Markers (CD44+/CD24− and ALDH1+): Prognostic Implications in Invasive Breast Cancer

BACKGROUND: Androgen receptor (AR) has emerged as a significant prognostic marker in early breast cancer (BCa). Association of AR with cancer stem cell (CSC) markers in BCa is unknown. Aim of the present study was to evaluate the immunohistochemical expression of AR, CD44, CD24 and ALDH1 in a cohort of Pakistani patients diagnosed with invasive BCa and to correlate the expression with 5- year disease free survival. PATIENTS AND METHODS: We evaluated immunohistochemical expression AR, CD44, CD24 and ALDH1 in formalin fixed paraffin embedded archival blocks of 166 cases of primary invasive BCa (stage I-III) and correlated the expression with clinicopathological variables and outcome using univariable and multivariable analysis. Survival data was computed by Kaplan Meier curves. RESULTS: Expression of AR was observed in 62.7% tumors whereas CD44, CD24 and ALDH1 were expressed in 61.4%, 44% and 30.1% tumors, respectively. AR expression was significantly associated with T1-T2 tumors, lower grade, estrogen and progesterone receptor expression (P < .05) and remained an independent prognostic indicator in multivariable analysis (adjusted HR 0.33, 95% CI 0.13–0.81; P = .016). Significant association was observed between concordant expression of AR and CD24 (P = .001) with a favorable impact on survival (P = .007) whereas expression of CSC phenotypes (CD44⁺, CD44⁺/CD24^? and ALDH1⁺) did not correlate with adverse outcome (P > .05). However, AR expression retained the association with better prognosis even in patients whose tumors exhibited a CSC phenotype. CONCLUSIONS: Expression of AR and CD24 in stage I-III invasive BCa correlates with favorable clinicopathological features and delineates a subgroup of patients with better disease-free survival.     

Bacteria-organic matter coupling and its significance for oceanic carbon cycling

This paper synthesizes current ideas on the role of the microbial loop in carbon fluxes in the ocean and proposes some directions for future research. Organic matter flux into bacteria is highly variable, which can significantly influence the pathways of carbon flow in the ocean. A goal for future research is to elucidate the mechanistic bases of bacteria-organic matter coupling. This research should take into consideration the micrometer-scale distribution of bacteria and the composition, structure, and dynamics of the organic matter field in the bacterium's microhabitat. The ideas on the interactions of bacteria with the particulate organic phase need to be revised in view of recent findings of highly abundant, previously unknown particles ranging in size from nanometers to hundreds of micrometers. The hot-spots in the distribution of organic matter and remineralized nutrients can influence the rates as well as the direction of biogeochemical fluxes. Slow-to-degrade dissolved organic matter (DOM) may be produced because of loose bacteria-organic matter coupling resulting in DOM storage. Its use at a later time and place has profound implications for carbon fluxes and food web dynamics. A fundamental research need for the future is to understand the ecological interactions among the members of the microbial loop in an appropriate microhabitat context. While this goal was previously intractable, new molecular and optical techniques should make it possible to understand the biogeochemical activities of the microbial loop in terms of the ecology and evolution of pelagic microbial communities.