Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.     

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.     

Tauro-β-muricholic acid restricts bile acid-induced hepatocellular apoptosis by preserving the mitochondrial membrane potential

Purposeβ-Muricholic acid (βMCA) is a trihydroxylated bile acid that constitutes the major bile acid in rat and mouse. βMCA is more hydrophilic than ursodeoxycholic acid and has been evaluated for dissolution of cholesterol gallstones. Since it is unknown if βMCA has beneficial effects on hepatocyte cell death we determined the effect of tauro-βMCA (TβMCA) on apoptosis in vitro.MethodsHuman Ntcp-transfected HepG2 cells and primary hepatocytes from rat and mouse were incubated with the proapoptotic glycochenodeoxycholic acid (GCDCA) as well as the free fatty acid palmitate in the absence and presence of TβMCA. Apoptosis was quantified using caspase 3/7-assays and after Hoechst 33342 staining. The mitochondrial membrane potential (MMP) was measured fluorometrically using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyaniniodide). Immunoblotting was performed against the proapoptotic Bcl-2-protein Bax.ResultsIn Ntcp-HepG2 cells, GCDCA markedly increased apoptosis after 4 h. Co-incubation with TβMCA reduced apoptosis to 49% (p < 0.01 vs. GCDCA, each; n = 6). While GCDCA (100 μmol/L) reduced the MMP to 34% after 6 h, combination treatment with TβMCA restored the MMP to control levels at all time points (n = 4). TβMCA also restored breakdown of the MMP induced by palmitate. GCDCA induced a translocation of Bax from the cytosol to mitochondria that was inhibited by simultaneous treatment with TβMCA in eqimolar concentrations.ConclusionsTβMCA restricts hepatocellular apoptosis induced by low micromolar concentrations of GCDCA or palmitate via inhibition of Bax translocation to mitochondria and preservation of the MMP. Thus, further studies are warranted to evaluate a potential use of TβMCA in ameliorating liver injury in cholestasis.     

Evaluation of the relevance of the glassy state as stability criterion for freeze-dried bacteria by application of the Arrhenius and WLF model

The aim of this work was to describe the temperature dependence of microbial inactivation for several storage conditions and protective systems (lactose, trehalose and dextran) in relation to the physical state of the sample, i.e. the glassy or non-glassy state. The resulting inactivation rates k were described by applying two models, Arrhenius and Williams–Landel–Ferry (WLF), in order to evaluate the relevance of diffusional limitation as a protective mechanism. The application of the Arrhenius model revealed a significant decrease in activation energy E_a for storage conditions close to T_g. This finding is an indication that the protective effect of a surrounding glassy matrix can, at least, partly be ascribed to its inherent restricted diffusion and mobility. The application of the WLF model revealed that the temperature dependence of microbial inactivation above T_g is significantly weaker than predicted by the universal coefficients. Thus, it can be concluded that microbial inactivation is not directly linked with the mechanical relaxation behavior of the surrounding matrix as it was reported for viscosity and crystallization phenomena in case of disaccharide systems.     

Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.     

Effects of systemic versus local administration of corticosteroids on mucosal tolerance

Respiratory exposure to allergen induces T cell tolerance and protection against the development of airway hyperactivity in animal models of asthma. Whereas systemic administration of dexamethasone during the delivery of respiratory Ag has been suggested to prevent the development of mucosal tolerance, the effects of local administration of corticosteroids, first-line treatment for patients with bronchial asthma, on mucosal tolerance remain unknown. To analyze the effects of systemic versus local administration of different types of corticosteroids on the development of mucosal tolerance, mice were exposed to respiratory allergen to induce mucosal tolerance with or without systemic or intranasal application of different doses of dexamethasone or prednisolone. After the induction of mucosal tolerance, proliferation of T cells was inhibited in tolerized mice, whereas systemic applications of corticosteroids restored T cell proliferation and secretion of Th2 cytokines. In contrast, inhaled corticosteroids showed no effect on both T cell proliferation and cytokine secretion. In addition, mice systemically treated with corticosteroids showed an increased airway hyperactivity with a significant lung inflammation, but also an increased T effector cells/regulatory T cells ratio in the second lymphoid organs when compared with mice that receive corticosteroids by inhalation. These results demonstrate that local administration of corticosteroids has no effect on the development of immune tolerance in contrast to systemically applied corticosteroids. Furthermore, although different concentrations of corticosteroids are administered to patients, our results demonstrated that the route of administration rather than the doses affects the effect of corticosteroids on respiratory tolerance induction. Considering the broad application of corticosteroids in patients with allergic disease and asthma, the route of administration of steroid substances seems crucial in terms of treatment and potential side effects. These findings may help elucidate the apparently contradicting results of corticosteroid treatment in allergic diseases.     

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.     

STOICHIOMETRIC RESPONSES OF PHYTOPLANKTON SPECIES TO THE INTERACTIVE EFFECT OF NUTRIENT SUPPLY RATIOS AND GROWTH RATES1

Three species of phytoplankton, Rhodomonas sp., Phaeodactylum tricornutum Bohlin, and Isochrysis galbana Parke, were cultivated in semicontinuous culture to analyze the response of carbon (C):nitrogen (N):phosphorus (P) stoichiometry to the interactive effect of five N:P supply ratios and four growth rates (dilution rates). The relationship between cellular N and P quotas and growth rates fits well to both the Droop and Ågren’s functions for all species. We observed excess uptake of both N and P in the three species. N:P biomass ratios showed a significant positive relationship with N:P supply ratios across the entire range of growth rates, and N:P biomass ratios converged to an intermediate value independent of N:P supply ratios at higher growth rates. The effect of growth rates on N:P biomass ratios was positive at lower N:P supply ratios, but negative at higher N:P supply ratios for both Rhodomonas sp. and I. galbana, while for P. tricornutum this effect was negative at all N:P supply ratios. A significant interactive effect of N:P supply ratios and growth rates on N:P biomass ratios was found in both Rhodomonas sp. and P. tricornutum, but not in I. galbana. Our results suggest that Ågren’s functions may explain the underlying biochemical principle for the Droop model. The parameters in the Droop and Ågren’s functions can be useful indications of algal succession in the phytoplankton community in changing oceans.     

Follicular dendritic cells emerge from ubiquitous perivascular precursors

The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRβ(+)-derived cells abolished FDC, indicating that FDC originate from PDGFRβ(+) cells. Lymphotoxin-α-overexpressing prion protein (PrP)(+) kidneys developed PrP(+) FDC after transplantation into PrP(-) mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRβ(+) stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin β receptor (LTβR)(-) kidney capsules, differentiated into Mfge8(+)CD21/35(+)FcγRIIβ(+)PrP(+) FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRβ(+) FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.