Ulf Forssmann

Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity.

β‐Defensins are antibiotic peptides involved in host defense at the epithelial surface. Three human β‐defensins (hBDs)‐‐hBD‐1, hBD‐2, and hBD‐3‐‐have been identified so far. We have characterized a new member of the β‐defensin family, hBD‐4, based on screening of genomic sequences and subsequent functional analysis. In contrast to hBD‐1, hBD‐2, and hBD‐3, which are diffusely expressed throughout many organs, hBD‐4 mRNA expression is confined to testis, stomach, uterus, neutrophils, thyroid, lung, and kidney. hBD‐4 expression was up‐regulated by infection with gram‐positive and gram‐negative bacteria in human respiratory epithelial cells, and in response to phorbol 12‐myristate 13‐acetate, but not in response to other inflammatory factors that up‐regulate the expression of hBD‐2 or hBD‐3. Synthetic hBD‐4 exhibits a selective, salt‐sensitive spectrum of antimicrobial activity, and it represents one of the most active antimicrobial peptides against Pseudomonas aeruginosa (minimal inhibitory concentration: 4.1 μg/ml) known to date. This new defensin is chemotactic for human blood monocytes, but it is inactive on neutrophils and eosinophils. These findings demonstrate the existence of a family of β‐defensin genes with different functions against diverse classes of microorganisms, regulated by different stimuli, and specific signal pathways, and confirm the relevance of antimicrobial peptides in host defense.

Identification of a novel, multifunctional beta-defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction.

Abstract. Previous studies have shown the implication of β-defensins in host defense of the human body. The human β-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human β-defensin, called human β-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-α (TNF-α), hBD-3 expression is increased particularly after stimulation by interferon-γ. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human β-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.

CKβ8, a novel CC chemokine that predominantly acts on monocytes

We have studied the biological properties of a new human CC chemokine, CKβ8, consisting of 99 amino acids including six cysteines. CKβ8 mRNA transcripts were induced in monocytes by IL‐1β and, to a lesser extent, by IFNγ, and were detected in RNA extracted from normal human liver and gastrointestinal tract. CKβ8 is chemotactic for monocytes, but is inactive on IL‐2 conditioned T lymphocytes, eosinophils and neutrophils. Desensitization experiments indicate that CKβ8 and MIP‐1β completely share receptors on monocytes and that the CKβ8 receptor, which appears to differ from the known ones, is also recognized by MCP‐1, MCP‐2, MCP‐3, MCP‐4, MIP‐1α and RANTES.

Inhibition of CD26/Dipeptidyl Peptidase IV Enhances CCL11/Eotaxin-Mediated Recruitment of Eosinophils In Vivo

Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11(3–74). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.

Hemofiltrate CC chemokines with unique biochemical properties: HCC-1/CCL14a and HCC-2/CCL15

The hemofiltrate CC chemokines CCL14a (formerly HCC‐1), CCL14b (formerly HCC‐3), and CCL15 (formerly HCC‐2) are encoded by mono‐ as well as bicistronic transcripts from a tandem gene arrangement on human chromosome 17q11.2. The transcription and splicing into several mono‐ and bicistronic transcripts of this gene complex are unique for human genes. No corresponding mechanism is known in nonprimate mammalian species such as mice and rats. The extremely high concentration of CCL14a in human plasma is exceptional for chemokines and led to the identification of this chemokine. Several molecular forms of CCL14a have been isolated and investigated. The mature propeptide CCL14a(1–74) is a low‐affinity agonist of CCR1 which is converted to a high‐affinity agonist of CCR1 and CCR5 on proteolytic processing by serine proteases. In contrast, CCL15 is characterized using molecular forms deduced from the mRNA/cDNA and shown to activate cells via CCR1 and CCR3, also dependent on the amino‐terminal length. Hemofiltrate CC chemokines are chemoattractants for different types of leukocytes including monocytes, eosinophils, T cells, dendritic cells, and neutrophils. In this review, we emphasize the genomic organization, expression patterns, and biochemical properties of CCL14a, CCL14b, and CCL15. We report results of significance for the development of therapeutic strategies, especially concerning HIV infection and inflammatory diseases.

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.

RECTUS ABDOMINIS MUSCULOPERITONEAL FLAP FOR CLOSURE OF BLADDER DEFECT

We describe closure of a bladder defect with a rectus abdominis musculoperitoneal flap after partial resection of the bladder wall in a patient who had a jejunovesical and jejunovaginal fistula due to rectal cancer. CASE REPORT A 74-year-old woman presented with fecal discharge through the vagina and urethra. She had undergone anterior rectal with partial bladder and vaginal stump resection for a T4N1M0 rectal carcinoma 10 years earlier. She had also undergone multiple revision laparotomies for tumor recurrence. At presentation ingested fluids were immediately discharged through the vagina. Transvesical and transvaginal fistulography did not show any connection to the small bowel. However, amylase in the vaginal discharge was 21,620 IU./l., and so the clinical diagnosis was jejunovesical and jejunovaginal fistula. At revision laparotomy a diffuse peritoneal carcinosis and frozen pelvis were found. The first and second jejunal loops were attached to the bladder and vagina, and there was a small fistula in a 4 3 2 cm. tumor between the jejunum and bladder/vagina. Jejunal segmentectomy was performed. However, the bladder could not be closed directly due to lateral fixation of the bladder wall to the tumor. Hence, a 5 3 9 cm. vertical rectus abdominis musculoperitoneal flap including the peritoneum was elevated. The flap was placed in the bladder defect with the peritoneum toward the lumen (see figure). Transuretheral and suprapubic catheters were removed at 1 week and 2 weeks postoperatively, respectively. The patient voided spontaneously thereafter.

Pharmacokinetic and Pharmacodynamic Characteristics of Subcutaneously Applied PTH-1-37

Background/Aims: Parathyroid hormone (PTH) derivatives exert pronounced renal and osteoanabolic properties when given intermittently. The current study was performed to assess the pharmacokinetic and pharmacodynamic properties as well as safety of subcutaneously applied PTH-1-37 after repeated dosing in healthy subjects. Methods: This randomized, double-blind, dose-escalating, placebo and active comparator controlled study was conducted in 33 healthy postmenopausal women. Subjects were allocated to one of five treatment options: 10, 20, or 40 µg PTH-1-37, 20 µg PTH-1-34 or placebo, administered as once daily subcutaneous doses for three days. Plasma drug concentrations and serum levels of endogenous PTH-1-84, and calcium as markers of biological activity were monitored during the treatment. Results: PTH was absorbed rapidly from the subcutaneous tissue with a median tmax of 30 minutes for 20 and 40 µg of PTH-1-37. tmax was 45 minutes for 20 µg PTH-1-34. Elimination half-lives were estimated as 76 ± 34 min and 70 ± 13 min for 20 µg and 40 µg PTH-1-37 (mean ± SD), and 78 ± 34 for 20 µg PTH-1-34. Both PTH fragments (PTH-1-37 and PTH-1-34) increased serum calcium. For PTH-1-37 the effect on serum calcium was dose-dependent. Suppression of endogenous PTH-1-84 was seen after the application of both PTH-1-37 and PTH-1-34. During the study period, the subjects experienced no unexpected or serious adverse events. Conclusions: PTH-1-37 is rapidly absorbed after s.c. injection, has a short plasma elimination half-life, and does not accumulate during multiple dosing. Biological activity was demonstrated by rising serum calcium and decreasing endogenous PTH-1-84 in blood plasma. The study drugs were well tolerated and safe. Our investigation presents data that PTH-1-37 is an excellent drug candidate for intervening with syndromes of dysregulation of calcium metabolism.