Cascading Trophic Interactions from Fish to Bacteria and Nutrients after Reduced Sewage Loading: An 18-Year Study of a Shallow Hypertrophic Lake

The effects of major reductions in organic matter, total phosphorus (TP), and total nitrogen (TN) loading on the chemical environment, trophic structure, and dynamics of the hypertrophic, shallow Lake Søbygård were followed for 18 years. After the reduction in organic matter loading in 1976, the lake initially shifted from a summer clear-water state, most likely reflecting high grazing pressure by large Daphniaspecies, to a turbid state with extremely high summer mean chlorophyll a (up to 1400 μg L^{? 1}), high pH (up to 10.2), and low zooplankton grazing. Subsequently, a more variable state with periodically high grazing rates on phytoplankton and bacteria was established. Changes in zooplankton abundance and grazing could be attributed to variations in cyprinid abundance due to a fish kill (probably as a consequence of oxygen depletion) and pH-induced variations in fish recruitment and fry survival. The results suggest strong cascading effects of fish on the abundance and size of zooplankton and phytoplankton and on phytoplankton production. A comparatively weak cascading effect on ciliates and bacterioplankton is suggested. Due to high internal loading, only minor changes were observed in lake-water TP after a reduction in external TP loading of approximately 80% in 1982; net retention of TP was still negative 13 years after the loading reduction, despite a short hydraulic retention time of a few weeks. TN, however, decreased proportionally to the TN-loading reduction in 1987, suggesting a fast N equilibration. Only minor improvement in the environmental state of the lake has been observed. We suggest that another decade will be required before the lake is in equilibrium with present external P loading.     

Separate Developmental Programs for HLA-A and -B Cell Surface Expression during Differentiation from Embryonic Stem Cells to Lymphocytes,Adipocytes and Osteoblasts

A major problem of allogeneic stem cell therapy is immunologically mediated graft rejection. HLA class I A, B, and Cw antigens are crucial factors, but little is known of their respective expression on stem cells and their progenies. We have recently shown that locus-specific expression (HLA-A, but not -B) is seen on some multipotent stem cells, and this raises the question how this is in other stem cells and how it changes during differentiation. In this study, we have used flow cytometry to investigate the cell surface expression of HLA-A and -B on human embryonic stem cells (hESC), human hematopoietic stem cells (hHSC), human mesenchymal stem cells (hMSC) and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA-B though at lower levels. IFNγ induced HLA-A to very high levels on both hESC and hMSC and HLA-B on hMSC. Even on hESC, a low expression of HLA-B was achieved. Differentiation of hMSC to osteoblasts downregulated HLA-A expression (P = 0.017). Interestingly HLA class I on T lymphocytes differed between different compartments. Mature bone marrow CD4⁺ and CD8⁺ T cells expressed similar HLA-A and -B levels as hHSC, while in the peripheral blood they expressed significantly more HLA-B7 (P = 0.0007 and P = 0.004 for CD4⁺ and CD8⁺ T cells, respectively). Thus different HLA loci are differentially regulated during differentiation of stem cells.     

Imaging of Metastatic Lymph Nodes by X-ray Phase-Contrast Micro-Tomography

Invasive cancer causes a change in density in the affected tissue, which can be visualized by x-ray phase-contrast tomography. However, the diagnostic value of this method has so far not been investigated in detail. Therefore, the purpose of this study was, in a blinded manner, to investigate whether malignancy could be revealed by non-invasive x-ray phase-contrast tomography in lymph nodes from breast cancer patients. Seventeen formalin-fixed paraffin-embedded lymph nodes from 10 female patients (age range 37–83 years) diagnosed with invasive ductal carcinomas were analyzed by X-ray phase-contrast tomography. Ten lymph nodes had metastatic deposits and 7 were benign. The phase-contrast images were analyzed according to standards for conventional CT images looking for characteristics usually only visible by pathological examinations. Histopathology was used as reference. The result of this study was that the diagnostic sensitivity of the image analysis for detecting malignancy was 100% and the specificity was 87%. The positive predictive value was 91% for detecting malignancy and the negative predictive value was 100%. We conclude that x-ray phase-contrast imaging can accurately detect density variations to obtain information regarding lymph node involvement previously inaccessible with standard absorption x-ray imaging.     

CX3CL1/CX3CR1 and CCL2/CCR2 Chemokine/Chemokine Receptor Complex in Patients with AMD

Purpose

The chemokine receptors CX3CR1 and CCR2 have been implicated in the development of age-related macular degeneration (AMD). The evidence is mainly derived from experimental cell studies and murine models of AMD. The purpose of this study was to investigate the association between expression of CX3CR1 and CCR2 on different leukocyte subsets and AMD. Furthermore we measured the plasma levels of ligands CX3CL1 and CCL2.

Methods

Patients attending our department were asked to participate in the study. The diagnosis of AMD was based on clinical examination and multimodal imaging techniques. Chemokine plasma level and chemokine receptor expression were measured by flow-cytometry.

Results

A total of 150 participants were included. We found a significantly lower expression of CX3CR1 on CD8⁺ T cells in the neovascular AMD group compared to the control group (p = 0.04). We found a significant positive correlation between CCR2 and CX3CR1 expression on CD8⁺ cells (r = 0.727, p = 0.0001). We found no difference in plasma levels of CX3CL1 and CCL2 among the groups.

Conclusions

Our results show a down regulation of CX3CR1 on CD8⁺ cells; this correlated to a low expression of CCR2 on CD8⁺ cells. Further studies are needed to elucidate the possible role of this cell type in AMD development.     

Production of target-specific recombinant human polyclonal antibodies in mammalian cells

We describe the expression and consistent production of a first target-specific recombinant human polyclonal antibody. An anti-Rhesus D recombinant polyclonal antibody, Sym001, comprised of 25 unique human IgG1 antibodies, was produced by the novel Sympress expression technology. This strategy is based on site-specific integration of antibody genes in CHO cells, using the FRT/Flp-In recombinase system. This allows integration of the expression construct at the same genomic site in the host cells, thereby reducing genomic position effects. Different bioreactor batches of Sym001 displayed highly consistent manufacturing yield, antibody composition, binding potency, and functional activity. The results demonstrate that diverse recombinant human polyclonal antibody compositions can be reproducibly generated under conditions directly applicable to industrial manufacturing settings and present a first recombinant polyclonal antibody which could be used for treatment of hemolytic disease of the newborn and/or idiopathic thrombocytopenic purpura.     

No Association between Loss-of-Function Mutations in filaggrin and Diabetes,Cardiovascular Disease,and All-Cause Mortality

Background

Common loss-of-function mutations in the filaggrin gene (FLG) are a major predisposing risk factor for atopic disease due to reduced epidermal filaggrin protein levels. We previously observed an association between these mutations and type 2 diabetes and hypothesized that an inherited impairment of skin barrier functions could facilitate low-grade inflammation and hence increase the risk of diabetes and cardiovascular disease. We examined the association between loss-of-function mutations in FLG and diabetes, stroke, ischemic heart disease (IHD), and all-cause mortality in the general population.

Methods

The R501X and 2282del4 loss-of function mutations in FLG were genotyped in four Danish study populations including a total of 13373 adults aged 15-77 years. Two of the studies also genotyped the R2447X mutation. By linkage to Danish national central registers we obtained information for all participants on dates of diagnoses of diabetes, stroke, and IHD, as well as all-cause mortality. Data were analyzed by Cox proportional hazard models and combined by fixed effect meta-analyses.

Results

In meta-analyses combining the results from the four individual studies, carriage of loss-of-function mutations in FLG was not associated with incident diabetes (hazard ratio (HR) (95% confidence intervals (CI)) = 0.95 (0.73, 1.23), stroke (HR (95% CI) = 1.27 (0.97, 1.65), ischemic heart disease (HR (95%CI) = 0.92 (0.71, 1.19), and all-cause mortality (HR (95%CI) = 1.02 (0.83, 1.25)). Similar results were obtained when including prevalent cases in logistic regression models.

Conclusion

Our results suggest that loss-of-function mutations in FLG are not associated with type 2 diabetes, cardiovascular disease, and all-cause mortality. However, larger studies with longer follow-up are needed to exclude any associations.     

Structural basis for ligand binding and processivity in cellobiohydrolase Cel6A from Humicola insolens

The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7-1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.