Preclinical pilot study monitoring topical drug penetration and dermal bioavailability of a peptidase inhibitor from different galenic formulations into pig dermis, using cutaneous microdialysis.

BACKGROUND: Cutaneous microdialysis (CM) is an ex vivo technique that allows study of tissue chemistry, including bioavailability of actual tissue concentration of unbound drug in the interstitial fluid of the body. AIM: To test the penetration and dermal bioavailability of galenic formulations of the small-molecule IP10.C8, a dual-protease inhibitor of the dipeptidyl peptidase and aminopeptidase families. METHODS: Using CM, we tested the penetration and dermal bioavailability of IP10.C8 into the dermis and subcutis of pigs, and determined the tissue concentration of IP10.C8 enzymatically, using an enzyme activity assay (substrate Gly-Pro-pNA) and high performance liquid chromatography. RESULTS: Dermal bioavailability was enhanced by using microemulsion or the addition of the penetration enhancer oleic acid to a hydroxyethylcellulose (HEC) gel formulation. Dermal bioavailability was also enhanced when galenic formulations were prepared with higher pH (7.5 vs. 6.5) or higher drug concentration (5% vs. 1%) in HEC gel. CONCLUSION: It seems possible, using CM for topical skin penetration testing in anaesthetized domestic pigs, to test the bioavailability of newly designed drugs. However, the experimental time is limited due to the anaesthesia, and is dependent on drug recovery. Validation of this technique for routine use is challenging, and more experiments are needed to validate this preclinical set-up.

[Alternative materials and solutions in total knee arthroplasty for patients with metal allergy]. / Alternative Werkstoffe und Lösungen in der Knieendoprothetik für Patienten mit Metallallergie.

The annual number of total knee replacement implantations is rising continuously. A progressive cutaneous hypersensitivity rate against metallic materials in the population has been registered which can lead to an increase of allergy-induced reactions associated with implant loosening in the future although the correlation with an allergic cutaneous sensitisation has not been proven in all cases. On apparent allergy against metallic implant components different alternative solutions to standard endoprostheses should be taken into account for primary implantation or revision of total knee replacement, for example the application of implant components without metallic elements (e.g. ceramics), the use of non-allergic metallic implants, such as titanium or ZrNb alloys, or potential allergy-inducing metallic materials after masking the implant surface using a suitable coating. In the case of primary or revision surgery most patients with metal allergy are treated with a Ti(Nb)N-coated knee implant made of cobalt-chrome or titanium alloys in our hospital. Within an international multi-centre study we are currently implanting a newly developed knee endoprosthesis system with a ceramic femoral component as an alternative.

The main neutral aminopeptidase activity of human lymphoid tumour cell lines does not originate from the aminopeptidase N-(APN; CD13) gene.

Lymphocytes and related cell lines are predominantly CD13-negative, however, there are reports describing neutral aminopeptidase activity in or on these cells. The aim of this study was to answer the question, whether this activity originates from APN-gene expression. The total cellular activities (Ala-pNA hydrolysis) of lymphoid cell lines are up to 15 times higher than that of normal lymphocytes. Despite weak or lacking CD13 surface expression all lymphoid cell lines tested contain APNmRNA as quantified by competitive RT-PCR as well as low enzymatic activity in their particulate fractions. By isoelectric focusing two enzyme species with isoelectric points of 5.4 or between 3.5 to 4.8, respectively, were detected. To investigate whether these activities result from APN-gene we established transfectants lacking cellular APN expression of the CD13-positive histiocytic cell line U937 and the CD13-negative T-cell line H9. Studies on these transfectants proved (I) that the main neutral aminopeptidase activity expressed in lymphoid cells is definitively not related to APN and (II) that APN is also expressed in lymphoid cells, although on a low level only.

Phorbol ester-induced shedding of intercellular adhesion molecule-1 (ICAM-1) on erythroleukemic K 562 cells.

The effect of phorbol 12-myristate 13-acetate (PMA) on the expression and shedding of intercellular adhesion molecule-1 (ICAM-1) was investigated on the hematopoietic cell lines K 562 and U 937 using flow cytometry, fluorescence microscopy and ELISA technique. At low concentration of 1 nM, PMA stimulated the expression of ICAM-1 on the cell surface about 4-fold within 24 h, whereas a short-term treatment with 100 nM PMA led to the shedding of 35% of ICAM-1 from the surface of K 562 cells. The release of surface ICAM-1 was found on single cells by fluorescence microscopy to be a uniform process proceeding within 15 min. The shedding of ICAM-1 correlated with elevated levels of sICAM-1 in the supernatants of cultured cells. Also on K 562 cells stimulated by TNF-alpha, a PMA-induced release of ICAM-1 was observed in addition to the known spontaneous shedding. In contrast to the results with K 562 cells, no PMA-induced shedding of ICAM-1 was found on U 937 cells. This indicates a cell-specific process for K 562 cells. The PMA-mediated release of ICAM-1 from K 562 cells suggests that the shedding process does not only occur in parallel to the surface expression of ICAM-1, but may be controlled by particular mechanisms of down-regulation.

Regulation of angiotensin II receptor subtypes during atrial fibrillation in humans.

BACKGROUND: Previous studies have suggested that atrial fibrillation (AF) is associated with the activation of the atrial angiotensin system. However, it is not known whether the expression of angiotensin II receptors changes during AF. The purpose of this study was to determine the atrial expression of angiotensin II type 1 and type 2 receptors (AT(1)-R and AT(2)-R) in patients with AF. METHODS AND RESULTS: Atrial tissue samples from 30 patients undergoing open heart surgery were examined. Eleven patients had chronic persistent AF (> or =6 months; cAF), 8 patients had paroxysmal AF (pAF), and 11 patients were in sinus rhythm. AT(1)-R and AT(2)-R were localized in the atrial tissue by immunohistochemistry and quantified at the protein and mRNA level by Western blotting and quantitative polymerase chain reaction. Both types of AT-R were predominantly expressed in atrial myocytes in all groups. The amount of AT(1)-R was reduced to 34.9% during cAF (P<0.01) and to 51.7% during pAF (P<0.05) compared with patients in sinus rhythm. In contrast, AT(2)-R was increased during cAF (246%; P=NS) and pAF (505%; P<0.01). AT(1)-R/AT(2)-R mRNA content was similar in all groups. CONCLUSIONS: AF is associated with the down-regulation of atrial AT(1)-R and the up-regulation of AT(2)-R proteins. These findings may help define the pathophysiological role of the angiotensin system in the structural remodeling of the fibrillating atria.

Increased expression of extracellular signal-regulated kinase and angiotensin-converting enzyme in human atria during atrial fibrillation.

OBJECTIVES: The purpose of this study was to determine whether atrial expression of the extracellular signal-regulated kinases Erk1/Erk2 and of the angiotensin-converting enzyme (ACE) is altered in patients with atrial fibrillation (AF). BACKGROUND: Recent studies have demonstrated that atrial fibrosis can provide a pathophysiologic substrate for AF. However, the molecular mechanisms responsible for the development of atrial fibrosis are unclear. METHODS: Atrial tissue samples of 43 patients undergoing open heart surgery were examined. Seventeen patients had chronic persistent AF (> or =6 months; CAF), 8 patients had paroxysmal AF (PAF) and 18 patients had no history of AF. Erk expression was analyzed at the mRNA (quantitative reverse transcription polymerase chain reaction), the protein (immunoblot techniques) and atrial tissue (immunohistochemistry) levels. Erk-activating kinases (MEK1/2) and ACE were analyzed by immunoblot techniques. RESULTS: Increased amounts of Erk2-mRNA were found in patients with CAF (75 +/- 20 U vs. sinus rhythm: 31 +/- 25 U; p < 0.05). Activated Erk1/Erk2 and MEK1/2 were increased to more than 150% in patients with AF compared to patients with sinus rhythm. No differences between CAF and PAF were found. The expression of ACE was three-fold increased during CAF. Amounts of activated Erk1/Erk2 were reduced in patients treated with ACE inhibitors. Patients with AF showed an increased expression of Erk1/Erk2 in interstitial cells and marked atrial fibrosis. CONCLUSIONS: An ACE-dependent increase in the amounts of activated Erk1/Erk2 in atrial interstitial cells may contribute as a molecular mechanism for the development of atrial fibrosis in patients with AF. These findings may have important impact on the treatment of AF.

More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control.

In addition to the mechanisms inducing the expression and secretion of cytokines under distinct pathophysiological conditions, the fate of cytokines after secretion at sites of inflammation is a field of growing interest. Proteolysis has been suggested to be a fundamental mechanism of regulating the activities of various components of the cytokine network. Evidence grows that besides highly specific cytokine converting proteases such as interleukin-1beta-converting enzyme or tumor necrosis factor-converting enzyme, neutrophil-derived serine proteases are intimately involved in the modulation of the activities of cytokines and their receptors. Particularly at sites of inflammation, high amounts of the active serine proteases elastase, cathepsin G, and proteinase 3 are released from infiltrating polymorphonuclear cells in close temporal correlation to elevated levels of inflammatory cytokines, strongly indicating that these proteases are involved in the control of cytokine bioactivity and availability.

Membrane-bound proteindisulfide isomerase (PDI) is involved in regulation of surface expression of thiols and drug sensitivity of B-CLL cells.

The proteindisulfide isomerase (PDI), a multifunctional cytoplasmic enzyme with additional chaperone activity, has been shown recently, using monoclonal antibodies, to be located on the membrane of mature human B lymphocytes and B cell chronic lymphocytic leukemia (B-CLL) cells. Here, evidence is presented that this antigen exhibits catalytic activity as measured by the reductive degradation of insulin (release of A chain molecules) on intact B cells in patients suffering from B-CLL, as well as on JVM 13 cells (B-CLL cell line). More than 98% of these cells exhibited PDI activity which could be inhibited by bacitracin and also by monoclonal and polyclonal antibodies to PDI. Interestingly, surface PDI expression was strongly correlated in our study with protein-bound membrane SH groups. These surface protein thiols were specifically determined by using low concentrations of the chloromethyl-derivative based fluorescent probe 5-(and6)-(((4-chloromethyl)-benzoyl)amino)-tetramethyl-rhodamine (CMTMR) at low temperature in the presence of sodium azide in flow cytometry. The highest PDI and SH expression was found on B lymphocytes, particularly B-CLL cells. The mean fluorescence intensity (MFI) of CMTMR-positive B cells in the B-CLL line was up to 10-fold higher than that of controls, indicating a strong elevation of cell membrane-located protein thiols on malignant B cells. The link between PDI and SH expression on cell surfaces points to a functional interaction between the two. Treatment with bacitracin resulted in a strong inhibition of PDI and a dramatic increase in surface protein thiol expression of B-CLL cells. Similar effects could be observed by cell treatment with anti-PDI antibodies, indicating that this enzyme system plays a crucial role in the regulation of protein-bound SH groups. Interestingly, artificially induced protein thiol expression led to significantly higher cellular resistance to the cytostatic drugs chlorambucil, vinblastin, and cisplatin in vitro as measured by cell growth. These data suggest for the first time a regulatory effect of PDI on the surface protein thiol status of B cells. The increased expression of PDI may play a crucial role in SH-mediated protection and drug resistance in malignant B lymphocytes.

Dipeptidyl peptidase IV (CD 26) and aminopeptidase N (CD 13) catalyzed hydrolysis of cytokines and peptides with N-terminal cytokine sequences.

A number of natural cytokines are characterized as having dipeptidyl peptidase (DP) IV susceptible N-terminal peptide sequences. Here we demonstrate that oligopeptides with sequences analogous to the N-terminal part of human IL-1 beta, IL-2, TNF-beta and murine IL-6 were hydrolyzed by purified DP IV and aminopeptidase N (AP-N). The rate of DP IV-catalyzed hydrolysis of these peptides was negatively correlated with their chain length. In contrast to these results, no degradation was found under our conditions for the intact recombinant cytokines, IL-1 alpha, IL-1 beta, IL-2, G-CSF and for natural IL-2, independent of whether DP IV and AP-N were used separately or in combination.

Evidence for a crucial role of neutrophil-derived serine proteases in the inactivation of interleukin-6 at sites of inflammation.

The bioactivity of interleukin-6 (IL-6) was found to be dramatically reduced in fluids from sites of inflammation. Here, we provide evidence that the neutrophil-derived serine proteases elastase, proteinase 3 and cathepsin G are mainly involved in its degradation and subsequent inactivation. The initially hydrolyzed peptide bonds were detected to be Val(11)-Ala(12) and Leu(19)-Thr(20) (elastase), Phe(78)-Asn(79) (cathepsin G) and Ala(145)-Ser(146) (proteinase 3). The soluble IL-6 receptor elicits a protective effect against the IL-6 inactivation by cathepsin G only. The inactivation of IL-6 by neutrophil-derived serine proteases might act as a feedback mechanism terminating the IL-6-induced activation of neutrophils.

Quantification of aminopeptidase N mRNA in T cells by competitive PCR.

The aminopeptidase N (CD13, EC 3.4.11.2) is a well-characterized surface molecule expressed in a variety of cell types and species. Recent data indicate an expression of the APN mRNA and the corresponding aminopeptidase activity in human peripheral T cells and related cell lines as well. Here, the sensitive method of competitive PCR was used to quantify low amounts of APN mRNA in T cell lines. An APN cDNA fragment enshortened by a deletion of 87 bp was used as an internal APN-specific standard. The myelo-monocytic cell line U937 and the lymphoid T cell lines HuT78 and H9 contain 2.3 x 10(7), 5.9 x 10(6) and 5.6 x 10(6) copies/micrograms total RNA, corresponding to 160, 70 and 50 copies/cell, respectively. These data have been confirmed by determination of the APN activity, that represents a fraction only of the total cellular neutral aminopeptidase activity in hematopoetic cells. In the case of the CD13-positive cell line U937, approximately 60-70% of the total neutral aminopeptidase activity could be attributed to APN. In contrast, only a minor fraction (5-20%) of the cellular neutral aminopeptidase activity in the T cell lines H9 and HuT78 represents APN. The results suggest that APN gene expression within the hematopoetic system is not restricted to myelo-monocytic cells, instead a low APN expression may be a common feature of lymphocytes, at least of T cells, too.

Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV).

Increased concentrations of procalcitonin (PCT) are found in the plasma of patients with thermal injury and in patients with sepsis and severe infection, making this molecule important as a diagnostic and prognostic marker in these diseases. Interestingly, only the truncated form of PCT, PCT(3-116), is present in the plasma of these patients. The enzyme responsible for this truncation is unknown as yet. Here, using capillary zone electrophoresis, mass spectrometry and Edman sequence analysis, we demonstrate that dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) is capable of catalyzing the hydrolysis of PCT(1-116), releasing the N-terminal dipeptide Ala-Pro. We hypothesize that PCT(3-116) is the result of the hydrolysis of PCT(1-116) by soluble DP IV of the blood plasma or by DP IV expressed on the surface of cells.

The activation-dependent induction of APN-(CD13) in T-cells is controlled at different levels of gene expression.

Recently, it was shown that aminopeptidase N (E.C. 3.4.11.2, CD13) is up-regulated during mitogenic stimulation of peripheral T-cells. In this study, we demonstrate that the half-life of APN mRNA was considerably prolonged in these cells leading to a 2.7-fold increase of APN transcript level. The apparent half-life time of the APN transcript was investigated by the RNA synthesis inhibitor-chase method using actinomycin D. The steady-state APN mRNA levels was determined by a competitive RT-PCR. The half-lives estimated in resting T-cells, natural killer cells and permanently growing tumour cells varied between 3.5 and 6 h. Finally, nuclear run-on assays revealed that the APN gene expression of stimulated T-cells is controlled by increased promoter activity as well. These studies suggest a control of APN gene expression at the post-transcriptional level in addition to promoter-mediated regulation.

R-esp1, a rat homologue of drosophila groucho, is differentially expressed after optic nerve crush and mediates NGF-induced survival of PC12 cells.

The differential display reverse transcription polymerase chain reaction method was used to detect alterations in gene expression in the superior colliculus after optic nerve crush in adult rats. One of the most prominent changes observed was the selective induction of R-esp1, a homologue of the Drosophila enhancer of split locus (Groucho). Therefore, we studied the influence of R-esp1 on nerve growth factor (NGF)-induced cell survival of PC12 cells. Overexpression of R-esp1 promotes cell survival even in the absence of NGF and, conversely, it is reduced by antisense-mediated inhibition of R-esp1 expression. In conclusion, we propose a novel model in which R-esp1 protein mediates the NGF-signaling pathway.

The N-terminal X-X-Pro sequence of the HIV-1 Tat protein is important for the inhibition of dipeptidyl peptidase IV (DP IV/CD26) and the suppression of mitogen-induced proliferation of human T cells.

Recent data in the literature suggest that the HIV-1 Tat(1-86) protein exhibits immunosuppressive effects. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV), which is identical to the T cell activation marker CD26. Here we show that the N-terminal amino acid sequence of Tat is essential for the inhibition of DP IV-catalyzed IL-2(1-12) degradation. N-terminal modification of Tat with rhodamine prevented inhibition of enzymatic activity of DP IV as well as suppression of DNA synthesis of mitogen-stimulated human T cells. Moreover, natural peptides containing the X-X-Pro N-terminal motif of Tat also inhibited DP IV activity. These data suggest the existence of endogenous immunomodulatory oligopeptides which influence immune cell proliferation and differentiation via DP IV as does HIV-1 Tat.

Evidence of a defective thiol status of alveolar macrophages from COPD patients and smokers. Chronic obstructive pulmonary disease.

In increasing numbers of pulmonary diseases an association with a loss of intracellular thiols, mainly glutathione, is postulated. Therefore, the quantitative measurement of thiols within different viable cells is a possible metabolic parameter for cellular function and defense capacity of all pulmonary immune cells including alveolar macrophages (AM), that are highly compromised by oxidative stress. In this study the cellular thiol content was determined using fluorochrom conjugated chloromethyl derivatives (5-chloromethylfluorescein diacetate, CMFDA) in flow cytometry. The procedure was evaluated in vitro using biochemical techniques for glutathione quantification. Based on this approach, AM obtained from bronchoalveolar lavage (BAL) of smokers and patients with chronic obstructive pulmonary disease (COPD) showed a significant thiol deficiency compared to a nonsmoker/non-COPD group. The cellular thiol expression of AM from smokers and COPD patients reached only 50 and 53% of the control group. Lowest thiol concentrations (47% of control) were detected within the smoker(+)/COPD(+) group. This intracellular thiol deficiency significantly correlated with reduced lung function (FEV(1), PaO(2)). With regard to the tightly regulated thiol metabolism of immune cells, these results imply the onset of functional disturbances in thiol deficient AM. The determination of the cellular thiol content of AM, obtained from BAL by flow cytometry, presents a simple and reliable tool to monitor the effect of therapeutic measures focusing on the stabilization of the cellular thiol status.

Dipeptidyl peptidase IV (DP IV, CD26) is involved in regulation of DNA synthesis in human keratinocytes.

Various studies have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK, and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. Here, we clearly demonstrate that this enzyme is highly expressed also on human epidermal foreskin and split-skin keratinocytes and that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit the enzymatic activity as well as the DNA synthesis of these cells. These data demonstrate that CD26 plays a role also in regulation of DNA synthesis of epidermal keratinocytes and that the enzymatic activity is required for mediating these effects.

Interleukin-6 and transforming growth factor-beta 1 control expression of cathepsins B and L in human lung epithelial cells.

Cathepsins B and L are commonly expressed cysteine proteinases that play a major role in lysosomal bulk proteolysis, protein processing, matrix degradation, and tissue remodeling. Cathepsins are also implicated in tumor progression and metastasis, tissue injury, and inflammation. Cells at sites of inflammation often show upregulation and secretion of cathepsins. The regulation of cathepsin expression by inflammatory mediators is not well understood. The aims of this study were to investigate the effect of the cytokines interleukin-1 beta (IL-1 beta), IL-6, IL-10, transforming growth factor-beta 1 (TGF-beta 1), and hepatocyte growth factor (HGF) on expression of cathepsin B and cathepsin L mRNA (quantitative RT-PCR), on protein expression (ELISA, Western blot), and also on enzymatic activity of cathepsins B and L. Investigations were performed using the human lung epithelial cell line A-549. IL-6 was found to induce a concentration-dependent increase in mRNA expression, protein concentration, and enzymatic activity of cathepsin L. Cathepsin B mRNA and protein expression were not affected by IL-6. In contrast, TGF-beta 1 decreased the amount of cathepsin L mRNA and cathepsin B mRNA. At protein level, it was shown that TGF-beta 1 clearly reduced the concentration of cathepsin L but not cathepsin B. The cytokines IL-1 beta, IL-10, and HGF were found to exert no effect on cathepsin B and L expression. In conclusion, these results are the first to show that IL-6 and TGF-beta 1 have opposite effects on the regulation of expression of cathepsins B and L in A-549 human lung epithelial cells. The proinflammatory cytokine IL-6 induced an upregulation of cathepsin L, whereas TGF-beta 1 suppressed cathepsin B and L expression. Further studies are needed to clarify the mechanism that affects cathepsin B and L expression.

Transforming growth factor beta 1 inhibits interleukin-10 mRNA expression and production in pokeweed mitogen-stimulated peripheral blood mononuclear cells and T cells.

The multifunctional cytokine transforming growth factor-beta 1 (TGF-beta 1) is known to inhibit the proliferation of lymphocytes. Whether this effect is a result of a direct action of TGF-beta 1 or an involvement of other "immunoinhibitory" cytokines is not yet clear. Here we have analyzed the effects of TGF-beta 1 on IL-10 and IL-1RA production in pokeweed mitogen-stimulated peripheral blood mononuclear cells (PBMC) and purified T lymphocytes. We show in these systems that TGF-beta 1 at a concentration of 10 ng/ml significantly suppresses both IL-10 mRNA expression and IL-10 production. IL-2 and IL-6 were capable of abolishing the effect of TGF-beta 1 on DNA synthesis and production of IL-10 by T lymphocytes in an additive manner. However, TGF-beta 1 did not influence IL-1RA production in PWM-stimulated PBMC. The present data show that the inhibitory effect of TGF-beta 1 on mitogen-activated immune cells is not the consequence of induction of the inhibitory cytokines IL-10 or IL-1RA but rather an inhibitory action on the production of IL-2 and/or IL-6.

Effect of granulocyte colony-stimulating factor treatment on ex vivo neutrophil functions in nonneutropenic surgical intensive care patients.

Granulocyte colony-stimulating factor (G-CSF) preferentially stimulates growth and differentiation of neutrophil precursors and activates neutrophil functions. The aim of the present study was to investigate the functional response of the neutrophil to exogenous recombinant human G-CSF (rHuG-CSF) in nonneutropenic patients. In 30 surgical intensive care unit patients with severely impaired wound healing, leukocyte differential count, plasma G-CSF level, and a broad spectrum of neutrophil functions were monitored before (day 0), throughout (days 1 and 5), and at days 1 and 5 after stopping G-CSF treatment. G-CSF application resulted in a 3.5-fold increase in peripheral blood granulocyte count at day 5 of treatment. The mean plasma G-CSF level rose from 48 to a maximum of 2314 pg/ml at day 1 of G-CSF therapy. Neutrophil chemotaxis and stimulated lysozyme release were decreased throughout G-CSF treatment, whereas respiratory burst activity, phagocytic activity, and intracellular calcium concentration were enhanced by G-CSF. Neutrophil membrane depolarization remained unaffected. The increased count and activation state of neutrophils were associated with clinical improvement in most of these patients. Thus, G-CSF may be a useful adjuvant treatment for nonneutropenic patients with severely impaired wound healing.