Rudolf Richter

Composition of the peptide fraction in human blood plasma: database of circulating human peptides

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, beta2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aalpha-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.

Human cord blood stem cells generate human cytokeratin 18-negative hepatocyte-like cells in injured mouse liver

Differentiation of adult bone marrow (BM) cells into nonhematopoietic cells is a rare phenomenon. Several reports, however, suggest that human umbilical cord blood (hUCB)-derived cells give rise to hepatocytes after transplantation into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, we analyzed the hepatic differentiation potential of hUCB cells and compared the frequency of newly formed hepatocyte-like cells in the livers of recipient NOD-SCID mice after transplantation of hUCB versus murine BM cells. Mononuclear cell preparations of hUCB cells or murine BM from enhanced green fluorescent protein transgenic or wild-type mice were transplanted into sublethally irradiated NOD-SCID mice. Liver regeneration was induced by carbon tetrachloride injury with and without subsequent hepatocyte growth factor treatment. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, we detected clusters of hepatocyte-like cells in the livers of hUCB-transplanted mice. These cells expressed human albumin and Hep Par 1 but mouse CK18, suggesting the formation of chimeric hepatocyte-like cells. Native fluorescence microscopy and double immunofluorescence failed to detect single hepatocytes derived from transplanted enhanced green fluorescent protein-transgenic mouse BM. Fluorescent in situ hybridization rarely revealed donor-derived hepatocyte-like cells after cross-gender mouse BM transplantation. Thus, hUCB cells have differentiation capabilities different from murine BM cells after transplantation into NOD-SCID mice, demonstrating the importance of further testing before hUCB cells can be used therapeutically.

Peptide bank generated by large-scale preparation of circulating human peptides.

Human hemofiltrate (HF) is a source for the purification of circulating regulatory peptides. HF is obtained in large quantities during treatment of patients suffering from chronic renal failure. We have developed a large-scale method for separating peptides from amounts up to 10,000 1 HF into 300 fractions in a standardized two-step procedure, employing cation-exchange separation, followed by reversed-phase chromatography. These fractions represent a peptide bank containing bioactive, desalted and lyophilized peptides of blood. Screening for and isolation of regulatory human peptides is simplified by using this peptide bank.

Quantum Proteolytic Activation of Chemokine CCL15 by Neutrophil Granulocytes Modulates Mononuclear Cell Adhesiveness

Monocyte infiltration into inflammatory sites is generally preceded by neutrophils. We show here that neutrophils may support this process by activation of CCL15, a human chemokine circulating in blood plasma. Neutrophils were found to release CCL15 proteolytic activity in the course of hemofiltration of blood from renal insufficiency patients. Processing of CCL15 immunoreactivity (IR) in the pericellular space is suggested by a lack of proteolytic activity in blood and blood filtrate, but a shift of the retention time (tR) of CCL15-IR, detected by chromatographic separation of CCL15-IR in blood and hemofiltrate. CCL15 molecules with N-terminal deletions of 23 (Δ23) and 26 (Δ26) aa were identified as main proteolytic products in hemofiltrate. Neutrophil cathepsin G was identified as the principal protease to produce Δ23 and Δ26 CCL15. Also, elastase displays CCL15 proteolytic activity and produces a Δ21 isoform. Compared with full-length CCL15, Δ23 and Δ26 isoforms displayed a significantly increased potency to induce calcium fluxes and chemotactic activity on monocytes and to induce adhesiveness of mononuclear cells to fibronectin. Thus, our findings indicate that activation of monocytes by neutrophils is at least in part induced by quantum proteolytic processing of circulating or endothelium-bound CCL15 by neutrophil cathepsin G.

Discovery and characterization of an endogenous CXCR4 antagonist.

CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.

Detection of immunoreactive atrial and brain natriuretic peptides in the equine atrium

The distribution of immunoreactivity (IR) for cardiodilatin/atrial natriuretic peptide (CDD/ANP) and brain natriuretic peptide (BNP) was examined immunohistochemically and immuno-electron-microscopically in the equine atrium, using specific antibodies. In the immunohistochemical studies, IR-CDD/ANP and IR-pBNP-26 (porcine BNP-26 immunoreactivity) was detected in the cytoplasm of the auricular cardiocytes, but IR-hBNP-32 (human BNP-32 immunoreactivity) was not. The double immunogold labelling method for IR-hBNP-28 and IR-pBNP-26 revealed that gold particles of different sizes were located in the same secretory granules in the cardiocyte, but no gold particles for IR-hBNP-32 were detected. These results show that CDD/ANP and porcine BNP-like peptides are colocalized in the same secretory granules of the equine atrium. They suggest that the equine atrium secretes both CDD/ANP and BNP-like peptides.

Significance of N-Terminal Proteolysis of CCL14a to Activity on the Chemokine Receptors CCR1 and CCR5 and the Human Cytomegalovirus-Encoded Chemokine Receptor US28

The CC chemokine CCL14a is constitutively expressed in a large variety of tissues and its inactive proform CCL14a(1–74) circulates in high concentrations in plasma. CCL14a(1–74) is converted into CCL14a(9–74) by the proteases urokinase-type plasminogen activator and plasmin and is a highly active agonist for the chemokine receptors CCR1 and CCR5. In this study, a new CCL14a analog, CCL14a(12–74), was isolated from blood filtrate. To elucidate the functional role of the N terminus, a panel of N-terminally truncated CCL14a analogs were tested on the receptors CCR1 to CCR5 and on the human cytomegalovirus (HCMV)-encoded chemokine receptor US28. The rank order of binding affinity to these receptors and of the activation of CCR1 and CCR5-mediated intracellular Ca2+ concentration mobilization is CCL14a(6–74)<(7–74)<(8–74)≪(9–74) = (10–74)≫(11–74)≫(12–74). The almost identical affinities of CCL14a(7–74), CCL14a(9–74), and CCL14a(10–74) for the US28 receptor and the inhibition of US28-mediated HIV infection of 293T cells by all of the N-terminally truncated CCL14a analogs support the promiscuous nature of the viral chemokine receptor US28. In high concentrations, CCL14a(12–74) did reveal antagonistic activity on intracellular Ca2+ concentration mobilization in CCR1- and CCR5-transfected cells, which suggests that truncation of Tyr11 might be of significance for an efficient inactivation of CCL14a. A putative inactivation pathway of CCL14a(9–74) to CCL14a(12–74) may involve the dipeptidase CD26/dipeptidyl peptidase IV (DPPIV), which generates CCL14a(11–74), and the metalloprotease aminopeptidase N (CD13), which displays the capacity to generate CCL14a(12–74) from CCL14a(11–74). Our results suggest that the activity of CCL14a might be regulated by stringent proteolytic activation and inactivation steps.

Pretransplant human leukocyte antigen antibodies detected by single-antigen bead assay are a risk factor for long-term kidney graft loss even in the absence of donor-specific antibodies.

Clinical relevance of ELISA‐ and single‐antigen bead assay (SAB)‐detected pretransplant HLA antibodies (SAB‐HLA‐Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death‐censored graft loss was found in patients with pretransplant SAB‐HLA‐Ab [HR: 4.46; 95% confidence interval (CI): 1.47–13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB‐HLA‐Ab but without SAB‐detected donor‐specific Ab (SAB‐DSA) (HR: 4.91; 95% CI of 1.43–16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA‐DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long‐term graft loss in patients with pretransplant SAB‐HLA, even in the absence of DSA. SAB‐HLA‐Ab‐positive patients, being negative in ELISA or CDC assay, might profit from a well‐HLA‐DR‐matched graft and intensified immunosuppression.

Current Developments in Mobilization of Hematopoietic Stem and Progenitor Cells and Their Interaction with Niches in Bone Marrow

The clinical application of hematopoietic stem and progenitor cells (HSPCs) has evolved from a highly experimental stage in the 1980s to a currently clinically established treatment for more than 20,000 patients annually who suffer from hematological malignancies and other severe diseases. Studies in numerous murine models have demonstrated that HSPCs reside in distinct niches within the bone marrow environment. Whereas transplanted HSPCs travel through the bloodstream and home to sites of hematopoiesis, HSPCs can be mobilized from these niches into the blood either physiologically or induced by pharmaceutical drugs. Firstly, this review aims to give a synopsis of milestones defining niches and mobilization pathways for HSPCs, including the identification of several cell types involved such as osteoblasts, adventitial reticular cells, endothelial cells, monocytic cells, and granulocytic cells. The main factors that anchor HSPCs in the niche, and/or induce their quiescence are vascular cell adhesion molecule(VCAM)-1, CD44, hematopoietic growth factors, e.g. stem cell factor (SCF) and FLT3 Ligand, chemokines including CXCL12, growth-regulated protein beta and IL-8, proteases, peptides, and other chemical transmitters such as nucleotides. In the second part of the review, we revise the current understanding of HSPC mobilization. Here, we discuss which mechanisms found to be active in HSPC mobilization correspond to the mechanisms relevant for HSPC interaction with niche cells, but also deal with other mediators and signals that target individual cell types and receptors to mobilize HSPCs. A multitude of questions remain to be addressed for a better understanding of HSPC biology and its implications for therapy, including more comprehensive concepts for regulatory circuits such as calcium homeostasis and parathormone, metabolic regulation such as by leptin, the significance of autonomic nervous system, the consequences of alteration of niches in aged patients, or the identification of more easily accessible markers to better predict the efficiency of HSPC mobilization.

The novel allele HLA-DQB1*0636 of Caucasian origin has a unique amino acid exchange at position 186 of the beta 2 region.

The novel HLA-DQB1*0636 allele differs from HLA-DQB1*060401 in one nucleotide substitution at codon 186 in exon 3.