High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

The Fc glycosylation of therapeutic antibodies is crucial for their effector functions and their behavior in pharmacokinetics and pharmacodynamics. To monitor the Fc glycosylation in bioprocess development and characterization, high-throughput techniques for glycosylation analysis are needed. Here, we describe the development of a largely automated high-throughput glycosylation profiling method with multiplexing capillary-gel-electrophoresis (CGE) with laser induced fluorescence (LIF) detection using a DNA analyzer. After PNGaseF digestion, the released glycans were labeled with 9-aminopyrene-1,3,6-trisulfonic acid (APTS) in 96-well plates, which was followed by the simultaneous analysis of up to 48 samples. The peak assignment was conducted by HILIC-UPLC-MS/MS of the APTS-labeled glycans combined with peak fractionation and subsequent CGE-LIF analysis of the MS-characterized fractions. Quantitative data evaluation of the various IgG glycans was performed automatically using an in-house developed software solution. The excellent method accuracy and repeatability of the test system was verified by comparison with two UPLC-based methods for glycan analysis. Finally, the practical value of the developed method was demonstrated by analyzing the antibody glycosylation profiles from fermentation broths after small scale protein A purification.     

The structure of Mlc titration factor A (MtfA/YeeI) reveals a prototypical zinc metallopeptidase related to anthrax lethal factor

MtfA of Escherichia coli (formerly YeeI) was previously identified as a regulator of the phosphoenolpyruvate (PEP)-dependent:glucose phosphotransferase system. MtfA homolog proteins are highly conserved, especially among beta- and gammaproteobacteria. We determined the crystal structures of the full-length MtfA apoenzyme from Klebsiella pneumoniae and its complex with zinc (holoenzyme) at 2.2 and 1.95 Å, respectively. MtfA contains a conserved H¹⁴⁹E¹⁵⁰XXH¹⁵³+E²¹²+Y²⁰⁵ metallopeptidase motif. The presence of zinc in the active site induces significant conformational changes in the region around Tyr205 compared to the conformation of the apoenzyme. Additionally, the zinc-bound MtfA structure is in a self-inhibitory conformation where a region that was disordered in the unliganded structure is now observed in the active site and a nonproductive state of the enzyme is formed. MtfA is related to the catalytic domain of the anthrax lethal factor and the Mop protein involved in the virulence of Vibrio cholerae, with conservation in both overall structure and in the residues around the active site. These results clearly provide support for MtfA as a prototypical zinc metallopeptidase (gluzincin clan).     

Shifts in N and P Budgets of Heathland Ecosystems: Effects of Management and Atmospheric Inputs

In the present study we analyzed the combined effects of management (grazing, mowing, prescribed burning, sod-cutting) and atmospheric deposition on N and P budgets of heathland ecosystems (Lüneburger Heide nature reserve; N Germany). We hypothesize that management measures such as grazing and mowing can accelerate a deposition-induced imbalance of N and P pools as a result of a disproportionally high output of P. We analyzed management and deposition affected input–output flows of N and P and related them to changes in the nutritional status of Calluna vulgaris 5 years after treatment application. We found that grazing and mowing caused the highest net loss of P due to high P concentrations in the aboveground biomass. In contrast, prescribed burning only slightly affected P pools, as P remained in the system due to ash deposition. Management-mediated effects on N and P pools were mirrored in the nutritional status of Calluna vulgaris: at the grazed and mown sites, the P content of current season’s shoots significantly decreased within 5 years after treatments, whereas the N content remained unchanged. We conclude that grazing and mowing can accelerate declining availability of P and, thus, accelerate a deposition-induced shift from N- to P-limited plant growth in the medium term. In the face of ongoing atmospheric N loads management schemes need to combine high- and low-intensity measures to maintain both a diverse structure and balanced nutrient budgets in the long term.     

Th cells act via two synergistic pathways to promote antiviral CD8+ T cell responses

The mechanisms of how Th cells promote CD8(+) T cell responses during viral infections are largely unknown. In this study, we unraveled the mechanisms of T cell help for CD8(+) T cell responses during vaccinia virus infection. Our results demonstrate that Th cells promote vaccinia virus-specific CD8(+) T cell responses via two interconnected synergistic pathways: First, CD40L expressed by activated CD4(+) T cells instructs dendritic cells to produce bioactive IL-12p70, which is directly sensed by Ag-specific CD8(+) T cells, resulting in increased IL-2Rα expression. Second, Th cells provide CD8(+) T cells with IL-2, thereby enhancing their survival. Thus, Th cells are at the center of an important communication loop with a central role for IL-2/IL-2R and bioactive IL-12.     

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.     

The roles of receptor tyrosine kinases and their ligands in the wound repair process

Cutaneous wound repair is a tightly regulated and dynamic process involving blood clotting, inflammation, formation of new tissue, and tissue remodeling. Gene expression profiling of mouse and human wounds as well as first proteomics studies have identified a large number of genes and proteins that are up- or downregulated by skin injury, and some of them have been functionally characterized in animal models of wound repair. Among the key regulators of wound repair are growth factors, which control migration, proliferation, differentiation and survival of cells at different stages of the healing process. This review summarizes the results of functional studies performed in mammals that have identified important roles of receptor tyrosine kinases and their ligands in wound repair.     

Conjugated estrogens in the endometrium during the estrous cycle in pigs

Estrogen metabolism results in the formation of inactive estrogen sulphates and glucuronides. Despite the lack of receptor binding, circulating conjugated estrogens might serve as a reservoir for the active form through the involvement of specific cleaving enzymes. In order to elucidate the potential role that estrogen conjugates play in the regulation of the estrous cycle, we determined the concentration of progesterone, estrogen and estrogen conjugates in serum and endometrial homogenates of cycling gilts. In addition, we determined the mRNA expression changes of enzymes (UDP glucuronosyltransferase (UGT), β-glucuronidase (GUSB), sulphotransferases (SULT) and steroid sulphatase (STS)) and transporters (multidrug resistance-associated protein (MRP), organic anion-transporting polypeptide (OATPs)) involved in the estrogen metabolism in the endometrium across the estrous cycle. GUSB displayed highest expression at estrous (day 0), decreasing expression during metestrus (day 3 and 6), minimal expression on day 10 and 12, and increasing expression towards proestrus (day 18), suggesting either a stimulation by estrogens or a negative impact of progesterone. The mRNA expression of the influx-transporter OATP1A2 significantly increased from day 0 to 6 and decreased again by day 10, while the efflux-transporters (MRP1, MRP2, and MDR1) displayed minimal expression at day 3 and 6. The mRNA expression of the UDP-glucuronsyltransferases followed a similar pattern, with minimal expression found at day 6. The analyses of the concentration of local and circulating steroid hormones points towards an interaction of the analyzed transporters and enzymes with steroid hormones, thereby possibly regulating the reservoir of active steroids contributing to the endometrial function.     

Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis

Fibroblast growth factors (FGFs) are master regulators of organogenesis and tissue homeostasis. In this study, we used different combinations of FGF receptor (FGFR)-deficient mice to unravel their functions in the skin. Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. We identified loss of FGF-induced expression of tight junction components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier causes activation of keratinocytes and epidermal γδ T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiate an inflammatory response and induce a double paracrine loop through production of keratinocyte mitogens by dermal cells. Our results identify essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlight the importance of stromal–epithelial interactions in skin homeostasis and disease.     

Small-scale habitat use of black grouse (Tetrao tetrix L.) and rock ptarmigan (Lagopus muta helvetica Thienemann) in the Austrian Alps

We investigated the small-scale habitat use of two grouse species, black grouse (Tetrao tetrix L.) and rock ptarmigan (Lagopus muta helvetica Thienemann) in a study area in the Austrian Central Alps in summer. To build habitat suitability models, we applied multiple logistic regression using presence–absence data from fieldwork as the response variable and a set of habitat characteristics as explanatory variables, respectively. To gain a better understanding of the mechanisms that drive habitat selection, we tested for two-way interaction terms before excluding any variables from the initial variable set. Four explanatory variables significantly contributed to the black grouse model: dwarf shrub cover, dwarf shrub height, patchiness and ant hills. The final model for rock ptarmigan contained three explanatory variables: dwarf shrub cover, rock cover and dwarf shrub height. Most notably, the interaction terms dwarf shrub cover × patchiness in the black grouse model and dwarf shrub cover × dwarf shrub height, rock cover × dwarf shrub height in the rock ptarmigan model point out trade-off mechanisms between food, cover and overview providing features. Thus, our models do not only identify the parameters that mainly drive habitat selection, but also deepen our understanding about the causal relationships between these factors. Therefore, the information gained in this study allows for a deduction of appropriate habitat management strategies and supports conservation efforts of local stakeholders.