The cardiovascular hormone ANP interferes with LPS-induced early inflammatory pathways in vitro and in vivo

Altogether, we have proven that ANP mediated effects are diverse yet similar in different organs derived from endotoxemic mice and in isolated blood leukocytes. These ANP mediated interactions are proceeding at the onset of LPS-induced inflammation and sepsis. Primarily, we can clearly demonstrate that ANP preconditioning in endotoxemic mice yields TNF-alpha m-RNA reduction, determined in the spleen as well as in the liver. As investigated in whole liver tissue, ANP preconditioning mediates its beneficial effects by reducing LPS-induced transcription factor NF-kappaB activation. This reduction is caused by decreased phosphorylation of the NF-kappaB inhibitory factor IkappaBalpha, proximately leading to impaired degradation of IkappaBalpha protein. Thus, enhanced IkappaBalpha protein level in the cytosol prevent NF-kappaB translocation into the nucleus, and subsequently transcription factor activity and gene expression. These effects might be caused by or lead to the reduction in TNF-alpha gene expression, finally preventing liver failure. Secondly, besides the transcriptional regulation of TNF-aplha gene expression determined in spleen and liver tissue, we focused on ANP mediated effects in LPS stimulated murine and human blood derived leukocytes. Following LPS stimulation, we observed reduced total TNF-alpha protein levels as well as decreased TNF-alpha amounts on the cell-surface in ANP preconditioned blood leukocytes, respectively monocytes and neutrophils. These initial investigations indicate that the reduced TNF-alpha protein levels in leukocytes might either be evoked by interference of ANP in transcriptional or posttranscriptional processes. Eventually, due to its effects on key events of cell activation, such as the reduction of LPS-induced TNF-alpha expression, ANP may represent a promising beneficial autocrine substance in modulating early inflammatory signaling pathways

Atrial Natriuretic Peptide, a Regulator of Nuclear Factor-κB Activation in Vivo

Natriuretic peptides (NPs) comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis. Along this line, atrial NP (ANP) (international non-proprietary name: carperitide, HANP) is an approved drug for the treatment of acute heart failure. In recent years, evidence has been given that the NP system possesses a far broader biological spectrum than the regulation of blood pressure and volume homeostasis. In fact, a substantial amount of in vitro work indicates that ANP affects important inflammatory processes and signaling pathways. Quite surprisingly, however, no information exists on the in vivo antiinflammatory potential and signaling of ANP. We show here that pretreatment of lipopolysaccharide (Salmonella abortus equi, 2.5 mg/kg)-challenged mice with ANP (5μg/kg iv, 15 min) rapidly inhibits nuclear factor-κB activation via inhibition of phosphorylation and degradation of the IκB-α protein. ANP also reduces Akt activation upon lipopolysaccharide injection. In ANP-pretreated mice, the increase of TNF-α serum concentration is markedly prevented; most importantly, the survival of these animals improved. These findings demonstrate both in vitro and in vivo an antiinflammatory profile of ANP that deserves to be further investigated in a therapeutic perspective

High Affinity, Developability and Functional Size: The Holy Grail of Combinatorial Antibody Library Generation

Since the initial description of phage display technology for the generation of human antibodies, a variety of selection methods has been developed. The most critical parameter for all in vitro-based approaches is the quality of the antibody library. Concurrent evolution of the libraries has allowed display and selection technologies to reveal their full potential. They come in different flavors, from naïve to fully synthetic and differ in terms of size, quality, method of preparation, framework and CDR composition. Early on, the focus has mainly been on affinities and thus on library size and diversity. Subsequently, the increased awareness of developability and cost of goods as important success factors has spurred efforts to generate libraries with improved biophysical properties and favorable production characteristics. More recently a major focus on reduction of unwanted side effects through reduced immunogenicity and improved overall biophysical behavior has led to a re-evaluation of library design