Beate Schacher

NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer–based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1′, P2′, P3, P4, and P2 positions were tested. This enabled us to construct an α1-proteinase inhibitor variant (Ile-Lys-Pro-Arg−/−Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefèvre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils.

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles.

Background: A soluble form of IL-6 receptor mediates pathogenic IL-6 trans-signaling. Results: ADAM10 and ADAM17 release IL-6 receptor from both human and murine monocytes/macrophages, whereas in the blood IL-6 receptor is also present on microvesicles. Conclusion: Shedding of endogenous IL-6 receptor is similar in humans and mice. Significance: Microvesicle release represents a novel mode of soluble IL-6 receptor generation with potential clinical implications. Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.

Functional Cathepsin C mutations cause different Papillon–Lefèvre syndrome phenotypes

AIM The autosomal-recessive Papillon-Lefèvre syndrome (PLS) is characterized by severe aggressive periodontitis, combined with palmoplantar hyperkeratosis, and is caused by mutations in the Cathepsin C (CTSC) gene. This study aimed to identify CTSC mutations in different PLS phenotypes, including atypical forms and isolated pre-pubertal aggressive periodontitis (PAP). MATERIAL AND METHODS Thirteen families with different phenotypes were analysed by direct sequencing of the entire coding region and the regulatory regions of CTSC. The function of novel mutations was tested with enzyme activity measurements. RESULTS In 11 of 13 families, 12 different pathogenic CTSC mutations were found in 10 typical PLS patients, three atypical cases and one PAP patient. Out of four novel mutations, three result in protein truncation and are thus considered to be pathogenic. The homozygous c.854C>T nucleotide exchange (p.P285L) was associated with an almost complete loss of enzyme activity. The observed phenotypic heterogeneity could not be associated with specific genotypes. CONCLUSIONS The phenotypic variability of the PLS associated with an identical genetic background may reflect the influence of additional genetic or environmental factors on disease characteristics. CTSC mutation analyses should be considered for differential diagnosis in all children suffering from severe aggressive periodontitis.

Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis

BackgroundLoss-of-function point mutations in the cathepsin C gene are the underlying genetic event in patients with Papillon-Lefèvre syndrome (PLS). PLS neutrophils lack serine protease activity essential for cathelicidin LL-37 generation from hCAP18 precursor.AimWe hypothesized that a local deficiency of LL-37 in the infected periodontium is mainly responsible for one of the clinical hallmark of PLS: severe periodontitis already in early childhood.MethodsTo confirm this effect, we compared the level of neutrophil-derived enzymes and antimicrobial peptides in gingival crevicular fluid (GCF) and saliva from PLS, aggressive and chronic periodontitis patients.ResultsAlthough neutrophil numbers in GCF were present at the same level in all periodontitis groups, LL-37 was totally absent in GCF from PLS patients despite the large amounts of its precursor, hCAP18. The absence of LL-37 in PLS patients coincided with the deficiency of both cathepsin C and protease 3 activities. The presence of other neutrophilic anti-microbial peptides in GCF from PLS patients, such as alpha-defensins, were comparable to that found in chronic periodontitis. In PLS microbial analysis revealed a high prevalence of Aggregatibacter actinomycetemcomitans infection. Most strains were susceptible to killing by LL-37.ConclusionsCollectively, these findings imply that the lack of protease 3 activation by dysfunctional cathepsin C in PLS patients leads to the deficit of antimicrobial and immunomodulatory functions of LL-37 in the gingiva, allowing for infection with A. actinomycetemcomitans and the development of severe periodontal disease.

Degree of gingivitis correlates to systemic inflammation parameters

BACKGROUND Investigation of interrelations between periodontal and systemic inflammatory parameters in periodontal health. METHODS 40 periodontally healthy (probing pocket depths [PPD]<3.6 mm and from 3.6 mm to 4 mm without bleeding on probing [BOP]; up to 2 sites with a PPD from 3.6 mm to 5 mm and BOP or up to 4 sites with a PPD from 3.6 mm to 5 mm without BOP were accepted; BOP<or=14%) probands 23 to 44 years of age without any known actual infectious or inflammatory diseases were examined. Clinical parameters and blood samples were obtained. The blood was analyzed for C-reactive protein (CRP), elastase, and leukocyte counts. Regression models were calculated to explain the variation of the dependent variables serum CRP, elastase, and leukocytes (independent variables: sex, age, PCR, BOP, smoking, PPD). RESULTS The sample was characterized as: GBI: 2.9+/-2.0%; PCR: 14.9+/-8.2%; PPD: 2.0+/-0.2 mm; AL: 0.4+/-0.3 mm; BOP: 7.0+/-1.9%; leukocytes: 6.2+/-1.1/nl; (median/interquartile range) CRP: 0.062/0.04-0.107) mg/dl; elastase: 9.075/7.375-12.2 ng/ml. Inflammatory parameters were influenced by the following factors: CRP: female (p=0.008), PPD (p=0.15); elastase: female (p=0.002), PPD (p=0.005), BOP (p=0.141); leukocytes: female (p=0.061), pack years (p=0.061), PCR (p=0.082). CONCLUSIONS The levels of all investigated systemic inflammation parameters were higher in females than in males. Both serum CRP and elastase correlated even in periodontally healthy with mean PPD.

Regenerative therapy of infrabony defects with or without systemic doxycycline. A randomized placebo‐controlled trial

AIM Comparison of regenerative therapy of infrabony defects with and without administration of postsurgical systemic doxycycline (DOXY). METHODS In each of 61 patients one infrabony defect was treated with enamel matrix derivative (EMD), EMD plus filler or membrane at two centres. By random assignment patients received either 200 mg DOXY per day or placebo (PLAC) for 7 days after surgery. Prior to and 6 months after surgery probing pocket depths (PPD) and vertical attachment level (PAL-V) were obtained. RESULTS Fifty-four patients (DOXY: 27; PLAC: 27) were re-examined after 6 months and had been treated exclusively with EMD. Seven to 8 days after surgery 81% of defects in both groups showed complete flap closure. In both groups significant (p < 0.001) PPD reduction (DOXY: 3.87 ± 1.44 mm; PLAC: 3.67 ± 1.30 mm) and PAL-V gain (DOXY: 3.11 ± 1.50 mm; PLAC: 3.32 ± 1.83 mm) were observed. However, the differences failed to be statistically significant (PPD: 0.20; p = 0.588; PAL-V: 0.21; p = 0.657). CONCLUSIONS Two hundred milligram systemic DOXY administered for 7 days after therapy of infrabony defects with EMD failed to result in better PPD reduction and PAL-V gain compared with PLAC which may be due to low power (50%) and, thus, random chance.

Evaluation of two siblings with Papillon-Lefèvre syndrome 5 years after treatment of periodontitis in primary and mixed dentition.

BACKGROUND This case report describes the clinical and microbiologic long-term outcome 5 years after periodontal therapy of two siblings diagnosed with Papillon-Lefèvre syndrome (PLS) and tinea capitis. METHODS In 2005, two brothers diagnosed with PLS and tinea capitis began periodontal treatment. Both of them showed premature mobility of the primary dentition, markedly increased probing depths, and subgingival Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans; Aa). Initial therapy consisted of scaling and root planing based on the concept of full-mouth disinfection, extraction of periodontally hopeless deciduous teeth, and systemic antibiotics. Reevaluation of clinical parameters revealed a dramatic improvement. After that, the patients were enrolled in a stringent maintenance program. Microbiologic monitoring was performed 1 and 5 years after treatment. RESULTS Five years after initial treatment, the periodontal situation was stable in both patients. Residual deciduous teeth, with the exception of one tooth, could be retained and no further teeth were lost. Further disease progression on the previously involved teeth was controlled, and development of periodontitis on erupting teeth was prevented for a period of 5 years. CONCLUSIONS Even periodontally affected deciduous teeth can be treated successfully in patients with PLS. Suppression of Aa and a stringent maintenance program are of high importance.

Enamel Matrix Derivative in Propylene Glycol Alginate for Treatment of Infrabony Defects With or Without Systemic Doxycycline: 12- and 24-Month Results

BACKGROUND This aim of this study is to compare regenerative therapy of infrabony defects with and without administration of post-surgical systemic doxycycline (DOXY) 12 and 24 months after therapy. METHODS In each of 57 patients, one infrabony defect (depth ≥ 4 mm) was treated regeneratively using enamel matrix derivative at two centers (Frankfurt am Main and Heidelberg). By random assignment, patients received either 200 mg DOXY per day or placebo (PLAC) for 7 days after surgery. Twelve and 24 months after surgery, clinical parameters (probing depths [PDs] and vertical clinical attachment level [CAL-V]) and standardized radiographs were obtained. Missing data were managed according to the last observation carried forward. RESULTS Data of 57 patients (DOXY: 28; PLAC: 29) were analyzed (26 males and 31 females; mean age: 52 ± 10.2 years; 13 smokers). In both groups, significant (P <0.01) PD reduction (DOXY: 3.7 ± 2.2 mm; PLAC: 3.4 ± 1.7 mm), CAL-V gain (DOXY: 2.7 ± 1.9 mm; PLAC: 3.0 ± 1.9 mm), and bone fill (DOXY: 1.6 ± 2.7 mm; PLAC: 1.8 ± 3.0 mm) were observed 24 months after surgery. However, the differences between both groups failed to be statistically significant (PD: P = 0.574; CAL-V: P = 0.696; bone fill: P = 0.318). CONCLUSIONS Systemic DOXY, 200 mg/day for 7 days, after regenerative therapy of infrabony defects did not result in better PD reduction, CAL-V gain, or radiographic bone fill compared with PLAC 12 and 24 months after surgery, which may be attributable to low power and, thus, random chance.

An extraordinary form of the Melkersson-Rosenthal syndrome successfully treated with the tumour necrosis factor-α blocker adalimumab

Melkersson-Rosenthal syndrome (MRS) is a rare granulomatous inflammatory disease characterised by the triad of orofacial oedema, facial nerve palsy and furrowed tongue. We describe the case of a 29-year-old patient suffering from an oligosymptomatic form of the disease with orofacial oedema, cobblestone pattern on the buccal mucosa and swelling of the tongue, accompanied by intermittent fatigue, influenza-like symptoms, intermittent tinnitus and acute hearing loss. An increase of several autoimmune-associated antibodies was also detected. Treatment with prednisolone, azathioprine or methotrexate failed to adequately control all symptoms in the long term. In the absence of a specific and well-established therapy for MRS, treatment with adalimumab was administered. Under adalimumab, total remission of all symptoms was achieved, indicating that tumour necrosis factor-α blockers are a promising therapeutic option for patients with Melkersson-Rosenthal syndrome.

Analysis of urinary cathepsin C for diagnosing Papillon–Lefèvre syndrome

Papillon–Lefèvre syndrome (PLS) (OMIM: 245000) is a rare disease characterized by severe periodontitis and palmoplantar keratoderma. It is caused by mutations in both alleles of the cathepsin C (CatC) gene CTSC that completely abrogate the proteolytic activity of this cysteine proteinase. Most often, a genetic analysis to enable early and rapid diagnosis of PLS is unaffordable or unavailable. In this study, we tested the hypothesis that active CatC is constitutively excreted and can be easily traced in the urine of normal subjects. If this is true, determining its absence in the urine of patients would be an early, simple, reliable, low‐cost and easy diagnostic technique. All 75 urine samples from healthy control subjects (aged 3 months to 80 years) contained proteolytically active CatC and its proform, as revealed by kinetic analysis and immunochemical detection. Of the urine samples of 31 patients with a PLS phenotype, 29 contained neither proteolytically active CatC nor the CatC antigen, so that the PLS diagnosis was confirmed. CatC was detected in the urine of the other two patients, and genetic analysis revealed no loss‐of‐function mutation in CTSC, indicating that they suffer from a PLS‐like condition but not from PLS. Screening for the absence of urinary CatC activity soon after birth and early treatment before the onset of PLS manifestations will help to prevent aggressive periodontitis and loss of many teeth, and should considerably improve the quality of life of PLS patients.