Persistent Autoantibody-Production by Intermediates between Short-and Long-Lived Plasma Cells in Inflamed Lymph Nodes of Experimental Epidermolysis Bullosa Acquisita

Autoantibodies are believed to be maintained by either the continuous generation of short-lived plasma cells in secondary lymphoid tissues or by long-lived plasma cells localized in bone marrow and spleen. Here, we show in a mouse model for the autoimmune blistering skin disease epidermolysis bullosa acquisita (EBA) that chronic autoantibody production can also be maintained in inflamed lymph nodes, by plasma cells exhibiting intermediate lifetimes. After EBA induction by immunization with a mCOL7c-GST-fusion protein, antigen-specific plasma cells and CD4 T cells were analyzed. Plasma cells were maintained for months in stable numbers in the draining lymph nodes, but not in spleen and bone marrow. In contrast, localization of mCOL7c-GST -specific CD4 T cells was not restricted to lymph nodes, indicating that availability of T cell help does not limit plasma cell localization to this site. BrdU-incorporation studies indicated that pathogenic mCOL7c- and non-pathogenic GST-specific plasma cells resemble intermediates between short-and long-lived plasma cells with half-lives of about 7 weeks. Immunization with mCOL7c-GST also yielded considerable numbers of plasma cells neither specific for mCOL7c- nor GST. These bystander-activated plasma cells exhibited much shorter half-lives and higher population turnover, suggesting that plasma cell lifetimes were only partly determined by the lymph node environment but also by the mode of activation. These results indicate that inflamed lymph nodes can harbor pathogenic plasma cells exhibiting distinct properties and hence may resemble a so far neglected site for chronic autoantibody production.     

Optimization of the differentiation of human preadipocytes in vitro

This study aimed at developing an optimal protocol for proliferation and differentiation of preadipocytes that is a prerequisite for constructing an ideal biohybrid composed of viable adipose precursor cells in a three-dimensional matrix. Such an implant could represent an adequate solution for correcting soft tissue defects, e.g., extensive deep burns or tumor resections. Preadipocytes were isolated from human subcutaneous adipose tissue samples and cultured in Dulbecco's modified eagle medium (DMEM)/Ham's F12 medium (F12) or OPTIMEM medium with or without the addition of human serum (hS) or fetal calf serum (FCS). The advantages of fibronectin-coated culture dishes for preadipocyte yield after isolation and differentiation were evaluated. After culture expansion, differentiation was induced by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of serum. The extent of differentiation was assayed by measuring the activity of glycerophosphate dehydrogenase as well as counting of differentiated versus undifferentiated cells. Our results show that fibronectin coating does not only strongly increase the yield of preadipocytes after isolation from adipose tissue but also significantly enhances differentiation of precursor cells to mature adipocytes. For optimal cell expansion, DMEM/F12 is more promoting than OPTIMEM and culturing with FCS shows a slightly better proliferation compared with hS supplementation. Differentiation, in contrast, is significantly improved when hS is used instead of FCS during proliferation. Our results smooth the way for autologous preadipocyte culturing and show that hS for preadipocyte culturing opens new and promising perspectives for adipose tissue engineering by optimizing in vitro expansion in cell culture and inducing substantial differentiation.     

Fusiogenic endogenous-retroviral syncytin-1 exerts anti-apoptotic functions in staurosporine-challenged CHO cells

Fusiogenic glycoprotein syncytin-1, expressed in human placenta, is a promising candidate for acquiring a basic knowledge of placental syncytialization. However, its cellular mode of action is unidentified. We investigated whether syncytin-1 may exert influence on apoptotic processes. Therefore, we incubated CHO cells after stable transfection with syncytin-1 (CHO-52) in the presence or absence of staurosporine (STS), a kinase inhibitor well characterized to induce apoptosis. When testing the phenotype of CHO-52 cells, we could demonstrate that the induction of apoptosis by STS was delayed over a period of up to 24 h. Furthermore, the cell death rate was decreased by approx 75% following transfection of syncytin-1 in CHO-52 compared to mock-treated cells. In detail, after 18h of incubation with 500 nM STS, 64 ± 2% of CHO-52 cells were viable compared to 16 ± 1% of CHO-mocks, after 24 h 43 ± 3% vs 5 ± 2%, respectively. CHO-52 cells exhibited a lower expression of active caspase 3 and anti-apoptotic Bcl-2 was found to be increased in CHO-52 cells at baseline and following STS treatment. Our study provides first evidence that syncytin-1 serves anti-apoptotic function under certain conditions. A lessened activation of caspase 3 and an increased expression of Bcl-2 are possible mechanisms.     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Gamma-aminobutyric acid type B receptors are constitutively internalized via the clathrin-dependent pathway and targeted to lysosomes for degradation

Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of gamma-aminobutyric acid, type B (GABAB) receptors. Therefore, we analyzed internalization of GABAB receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40-50% of cell surface receptors was detected. Stimulation of GABAB receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonist-induced internalization. The data suggest that GABAB receptors were endocytosed via the classical dynamin- and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABAB receptors via clathrin-coated pits, which resulted in lysosomal degradation of the receptors.     

Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS

Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.     

Reliability of a new virtual reality test to measure cervicocephalic kinaesthesia

The aim of this study was to investigate the cervicocephalic kinaesthesia of healthy subjects for gender and age effects and its reliability in a new virtual reality test procedure. 57 healthy subjects (30 male, 27 females; 18-64 years) were immersed into a virtual 3D scene via a headmounted display, which generated specific head movements. The joint repositioning error was determined in a static and dynamic test at the times T0, T1 (T0 + 10 minutes) and T2 (T0 + 24 hours). The intrasession reliability (T0-T1) and the intersession reliability (T0-T2) were analysed. In both tests no gender- or age-specific effects were found. In the overall group the means of the static test were 6.2°-6.9° and of the dynamic test were 4.5°-4.9°. The intratest difference in the static test was -0.16° and the intertest difference was 0.47°. The intratest difference in the dynamic test was 0.42° and the intertest difference was 0.37°. The static and dynamic test was reproducible in healthy subjects, with minor deviations, irrespective of gender and age. The smaller interindividual differences in the dynamic test could be beneficial in the comparison of healthy individuals and individuals with cervical spine disorders.     

Altering an artificial Gagpolnef polyprotein and mode of ENV co-administration affects the immunogenicity of a clade C HIV DNA vaccine

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.