MMP-7 and TIMP-1, news targets in predicting poor wound healing in apical periodontitis

Introduction: Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions. Methods: Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy. Results: Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus. Conclusions: MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development

Grandisin caused morphological changes larval and toxicity on Aedes aegypti

Dengue is a tropical disease caused by an arbovirus transmitted by Aedes aegypti. Since no effective vaccine is available for treating dengue, the present study focused on population vector control through investigating the use of the lignan grandisin, isolated from Piper solmsianum C. DC., Piperaceae, against the larvae of A. aegypti. Grandisin caused larval (L3) mortality at LC50 150 µg/mL. Histological analysis on A. aegypti larvae treated with grandisin (LC50 50 µg/mL) showed changes in the anterior-middle midgut, with intense tissue destruction and cell disorganization.

Periodontal treatment downregulates protease-activated receptor 2 in human gingival crevicular fluid cells

Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.

Analysen zur Expression proinflammatorischer Mediatoren in humanen Zellkulturmodellen und murinen Tiermodellen chronisch-inflammatorischer Erkrankungen

Die humane induzierbare NO-Synthase (iNOS) spielt bei zahlreichen Erkrankungen wie Asthma, Krebs und der rheumatoiden Arthritis eine entscheidende Rolle. Durch Fehlregulation der iNOS-Expression kommt es häufig zu massiven Gewebeschädigungen. Aus diesem Grund ist es wichtig die Mechanismen der Genregulation der iNOS-Expression zu verstehen. Bei Affinitätschromatographie-Analysen wurde das zytosolische PolyA-bindende Protein (PABP) als direkter Interaktionspartner der 3´UTR der humanen iNOS identifiziert. Weitere Bindungsanalysen konnten eine spezifische Bindestelle für PABP in der 5´UTR und zwei Bindestellen im AU-reichen Bereich der 3´UTR der humanen iNOS nachweisen. Eine siRNA-mediierte Herabregulation von PABP mit Hilfe der stabilen Expression spezifischer siRNAs in DLD-1 Zellen (siPABP Zellen) zeigte eine signifikant verringerte Expression der humanen iNOS und damit einhergehend eine verringerte NO-Produktion nach Zytokinstimulation. Promotoranalysen zeigten keine Veränderung der Induzierbarkeit des humanen 16 kb iNOS-Promotors in siPABP Zellen. RNA-Stabilitätsanalysen zeigten einen verstärkten Abbau der iNOS-mRNA in diesen Zellen, so dass davon auszugehen ist, dass die Regulation der humanen iNOS über die mRNA-Stabilität erfolgt. Reportergen-Analysen mit Plasmiden, welche die 5’ und/oder 3’UTR Sequenzen der humanen iNOS mit den identifizierten PABP-Bindestellen oder Mutationen in diesen Bindestellen enthielten, zeigten, dass PABP die iNOS-mRNA über die 5´UTR stabilisiert und anscheinend über die 3´UTR einen destabilisierenden Effekt auf die mRNA ausübt. Ebenfalls scheint PABP über die 3’UTR dieTranslation der iNOS mRNA zu hemmen. Die Ergebnisse dieser Arbeit zeigen, dass PABP, über seine allgemeinen Funktionen hinaus, eine spezifische Rolle in der Regulation der Expression der humanen iNOS einnimmt.rnDie rheumatoide Arthritis (RA) ist eine chronisch entzündliche Autoimmunerkrankung, welche überwiegend die peripheren Gelenke der Hände und Füße betrifft. Die aktuellen Therapiemöglichkeiten sind immer noch mit einer Vielzahl von Nebenwirkungen behaftet und führen nicht zur vollständigen Remission der Erkrankung, so dass die Entwicklung neuer Medikamente unerlässlich ist. In dieser Arbeit wurden die antiinflammatorischen Substanzen Gallielalacton (Gal) und Oxacyclododecindion (Oxa) im Mausmodell der kollagen-induzierten Arthritis (CIA) getestet. Leider waren beide Substanzen nicht in der Lage die Symptome der CIA zu vermindern, obwohl beide im Modell der LPS-induzierten akuten Entzündung die Expression proinflammatorischer Mediatoren senken konnten. Die Substanz S-Curvularin (SC) hat sich im CIA-Modell bereits bewährt und wurde in dieser Arbeit weiter untersucht. SC war in der Lage die Expression knorpel- und knochendestruktiver Markergene signifikant zu verrindern. rnIn der vorliegenden Arbeit wurden neue microRNAs identifiziert, die in der Pathogenese der CIA eine Dysregulation zeigen. Die Expression dieser microRNAs wurde von SC wieder auf das Normalniveau gebracht, so dass SC eine vielversprechende Substanz in der Therapie chronisch inflammatorische Erkrangungen sein könnte. Die neu identifizierten CIA-relevanten microRNAs könnten als neueRA-Marker oder als Zielstrukturen für neue Medikamente dienen.rn

Morphological and immunocytochemical characterization of feline odontoclastic resorptive lesions

Objective: Feline odontoclastic resorptive lesions (FORL) are common in domestic cats. The disease is painful, and leads to root resorption and replacement by bone, and tooth loss. The cause of this non-curable disease has not been established. The present study focused on the periodontal ligament (PL) in clinically and histologically healthy teeth and in teeth exhibiting various degrees of FORL. Methods: A total of 176 tissue blocks from 29 teeth was available for light and transmission electron microscopy. An antibody against osteopontin (OPN) was used for high-resolution immunocytochemistry, since this protein is involved in bone remodeling, cell adhesion, and inflammation. Results: A partial low cell number and an occasional translucent perivascular zone characterized the PL in healthy looking teeth. In teeth exhibiting small or initial FORL, the perivascular hyalinization was more pronounced, cell number was reduced, and widened channels connected the PL with the alveolar bone. In teeth exhibiting severe forms of FORL, the PL tissue was drastically affected. PL regions with massive tissue destruction were characterized by a lack of both cells and extracellular matrix, while other regions were replaced by bone marrow stroma. OPN labeling was restricted to the PL-facing matrix portions of bone and cementum. Conclusion: PL alterations seem to be a common feature in feline teeth. PL degeneration may initially be associated with vasculitis and appeared to increase with the severity of FORL. Since the PL did not recover and bone marrow stroma occupied the degenerated PL, ankylosis and replacement resorption are regarded as unavoidable sequelae.

Detection of perforin and granzyme B mRNA expressing cells in lichen sclerosus

Granzyme B and perforin messenger RNA (mRNA) expression has been shown to be a specific in vivo activation marker for cytotoxic cells. The aim of this study was to assess the contribution of cell-mediated cytotoxicity in the pathogenesis of lichen sclerosus. In situ hybridization and immunohistochemistry were performed on serial tissue sections of lesional skin biopsies and normal skin as control. Immunohistochemical staining showed that the cellular infiltrate of diseased skin consisted predominantly of T cells (CD3+) and some B cells (CD20+). Among T cells CD4+ and CD8+ cells were found in about equal numbers. In normal skin samples perforin and granzyme B mRNA expressing cells were only rarely found. In contrast, in biopsies from diseased skin a high percentage of infiltrating cells expressed mRNA for perforin and granzyme B. The perforin and granzyme B expressing cells were found in the dermal infiltrate and intraepidermally in close proximity to keratinocytes suggesting in situ activation of these cells. These findings provide evidence that cell-mediated cytotoxicity plays a significant role in tissue destruction in lichen sclerosus.

Detection of apoptosis in vivo using antibodies against caspase-induced neo-epitopes

Cell death induction by apoptosis is an important process in the maintenance of tissue homeostasis as well as tissue destruction during various pathological processes. Consequently, detection of apoptotic cells in situ represents an important technique to assess the extent and impact of cell death in the respective tissue. While scoring of apoptosis by histological assessment of apoptotic cells is still a widely used method, it is likely biased by sensitivity problems and observed-based variations. The availability of caspase-mediated neo-epitope-specific antibodies offers new tools for the detection of apoptosis in situ. Here, we discuss the use of immunohistochemical detection of cleaved caspase 3 and lamin A for the assessment of apoptotic cells in paraffin-embedded liver tissue. Furthermore, we evaluate the effect of tissue pretreatment and antigen retrieval on the sensitivity of apoptosis detection, background staining and maintenance of tissue morphology.

Periodontal disease progression in subjects with orofacial clefts over a 25-year follow-up period

AIMS: To assess rates of periodontal disease progression in subjects with cleft lip, alveolus and palate (CLAP) over a 25-year period without regular maintenance care in a specialist setting and to compare those with those of subjects without alveolar clefts, i.e. cleft lip (CL) or cleft palate (CP). MATERIAL AND METHODS: Ten subjects with CLAP and 10 subjects with CL/CP were examined in 1979, 1987, 1993 and 2004. Probing pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BoP) and plaque control record (PCR) scores were recorded in all 20 subjects. RESULTS: High plaque and BoP scores were recorded at all examinations in both groups. Over 25 years, a statistically significant loss of mean full-mouth CAL of 1.52 +/- 0.12 mm (SD) and 1.66 +/- 0.15 mm occurred in the CLAP and CL/CP group respectively (p<0.05). A statistically significant increase (p<0.05) in mean full-mouth PPD of 0.35 +/- 0.12 mm was observed in the CL/CP group, whereas only a trend for a mean full-mouth increase in PPD of 0.09 +/- 0.11 mm was observed in the CLAP group. In subjects with CLAP, a statistically significant increase (p<0.05) in PPD of 0.92 +/- 1.13 mm at cleft sites was observed compared with that of 0.17 +/- 0.76 mm at control sites. With respect to CAL, the loss at the corresponding sites amounted to 2.71 +/- 1.46 and to 2.27 +/- 1.62 mm, respectively (p=0.36). CONCLUSIONS: When stringent and well-defined supportive periodontal therapy was not provided, subjects with orofacial clefts were at high risk for periodontal disease progression. Over 25 years, alveolar cleft sites tended to have more periodontal tissue destruction compared with control sites.

Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.

Pathophysiology of Hidradenitis suppurativa

Only limited data are available about the precise mechanism leading to tissue inflammation and damage in patients with hidradenits suppurativa (HS). The central pathogenetic event in HS is the occlusion of the upper parts of the hair follicle leading to a perifollicular lympho-histiocytic inflammation. In early lesions, neutrophilic abscess formation and influx of mainly macrophages, monocytes and dendritic cells predominate. In chronic disease, the infiltrate expand with increased frequencies of B cells and plasma cells. In the inflammatory infiltrates toll like receptor 2 (TLR2) was highly expressed by infiltrating macrophages and dendritic cells indicating that stimulation of inflammatory cells by TLR2 activating microbial products may be important trigger factors in the chronic inflammatory process. Furthermore, the pro inflammatory cytokines IL-12 and IL-23 are abundantly expressed by macrophages infiltrating papillary and reticular dermis of HS skin. Both of these cytokines are believed to be important mediators in autoimmune tissue destruction and its blocking by biologics has been shown to be effective in the treatment of psoriasis. Especially IL-23 has been shown to be involved in the induction of a T helper cell subset producing IL-17, therefore, named Th17, which is distinct from the classical Th1/Th2 subsets. In chronic HS lesions IL-17-producing T helper cells were found to infiltrate the dermis. An overexpression of various other cytokines like IL-1beta, CYCL9 (MIG), IL-10 , IL-11 and BLC has been described in HS lesion whereas IL-20 and IL-22 have been shown to be down regulated. Similar to psoriasis also in HS the antimicrobial peptides beta defensin 2 and psoriasin are highly upregulated. This may at least in part explain the clinical finding that HS patients suffer only rarely from skin infections. Taken together the inflammatory reaction leading to HS are only poorly understood, but they show many similarity with other inflammatory reactions as e.g. in psoriasis.

MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis.

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

IMMUNE MODULATION OF THE MYCOBACTERIUM TUBERCULOSIS GRANULOMATOUS RESPONSE

Tuberculosis (TB) remains a major public health burden. The immunocompetant host responds to Mycobacterium tuberculosis (MTB) infection by the formation of granulomas, which initially prevent uncontrolled bacterial proliferation and dissemination. However, increasing evidence suggests that granuloma formation promotes persistence of the organism by physically separating infected cells from effector lymphocytes and by inducing a state of non-replicating persistence in the bacilli, making them resistant to the action of antibiotics. Additionally, immune-mediated tissue destruction likely facilitates disease transmission. The granulomatous response is in part due to mycobacterial glycolipid antigens. Therefore, studies were first undertaken to determine the innate mechanisms of mycobacterial cord factor trehalose-6’6-dimycolate (TDM) on granuloma formation. Investigations using knock-out mice suggest that TNF-a is involved in the initiation of the granulomatous response, complement factor C5a generates granuloma cohesiveness, and IL-6 is necessary for maintenance of an established granulomatous responses. Studies were next performed to determine the ability of lactoferrin to modulate the immune response and pathology to mycobacterial cord factor. Lactoferrin is an iron-binding glycoprotein with immunomodulatory properties that decrease tissue damage and promote Th1 responses. Mice challenged with TDM and treated with lactoferrin had decreased size and numbers of granulomas at the peak of the granulomatous response, accompanied by increased IL-10 and TGF-b production. Finally, the ability of lactoferrin to serve as a novel therapeutic for the treatment of TB was performed by aerosol challenging mice with MTB and treating them with lactoferrin added to the drinking water. Mice given tap water had lung log10 CFUs of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7) and mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Total lung inflammation in lactoferrin treated mice was significantly decreased, with fewer areas of macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. The lungs of lactoferrin treated mice had increased CD4+ IFN-g+ cells and IL-17 producing cells on ELISpot analysis. It is hypothesized that lactoferrin decreases bacterial burden during MTB infection by early induction of Th1 responses.

Longitudinal extension of myelomalacia by intramedullary and subdural hemorrhage in a canine model of spinal cord injury

BACKGROUND CONTEXT In canine intervertebral disc (IVD) extrusion, a spontaneous animal model of spinal cord injury, hemorrhage is a consistent finding. In rodent models, hemorrhage might be involved in secondary tissue destruction by biochemical mechanisms. PURPOSE This study aimed to investigate a causal association between the extents of intramedullary, subdural and epidural hemorrhage and the severity of spinal cord damage following IVD extrusion in dogs. STUDY DESIGN/SETTING A retrospective study using histologic spinal cord sections from 83 dogs euthanized following IVD extrusion was carried out. METHODS The degree of hemorrhage (intramedullary, subdural, epidural), the degree of spinal cord damage in the epicenter (white and gray matter), and the longitudinal extent of myelomalacia were graded. Associations between the extent of hemorrhage and the degree of spinal cord damage were evaluated statistically. RESULTS Intramedullary and subdural hemorrhages were significantly associated with the degree of white (p<.001/ p=.004) and gray (both p<.001) matter damage, and with the longitudinal extension of myelomalacia (p<.001/p=.005). Intriguingly, accumulation of hemorrhagic cord debris inside or dorsal to a distended and ruptured central canal in segments distant to the epicenter of the lesion was observed exhibiting a wave-like pattern on longitudinal assessment. The occurrence of this debris accumulation was associated with high degrees of tissue destruction (all p<.001). CONCLUSIONS Tissue liquefaction and increased intramedullary pressure associated with hemorrhage are involved in the progression of spinal cord destruction in a canine model of spinal cord injury and ascending or descending myelomalacia. Functional and dynamic studies are needed to investigate this concept further.

Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein ɛ-deficient mice

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein ɛ (C/EBPɛ) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBPɛ develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3–5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBPɛ is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.

Causalgia, pathological pain, and adrenergic receptors

Control of expression of molecular receptors for chemical messengers and modulation of these receptors’ activity are now established as ways to alter cellular reaction. This paper extends these mechanisms to the arena of pathological pain by presenting the hypothesis that increased expression of α-adrenergic receptors in primary afferent neurons is part of the etiology of pain in classical causalgia. It is argued that partial denervation by lesion of peripheral nerve or by tissue destruction induces a change in peripheral nociceptors, making them excitable by sympathetic activity and adrenergic substances. This excitation is mediated by α-adrenergic receptors and has a time course reminiscent of experimental denervation supersensitivity. The change in neuronal phenotype is demonstrable after lesions of mixed nerves or of the sympathetic postganglionic supply. Similar partial denervations also produce a substantial increase in the number of dorsal root ganglion neurons evidencing the presence of α-adrenergic receptors. The hypothesis proposes the increased presence of α-adrenergic receptors in primary afferent neurons to result from an altered gene expression triggered by cytokines/growth factors produced by disconnection of peripheral nerve fibers from their cell bodies. These additional adrenergic receptors are suggested to make nociceptors and other primary afferent neurons excitable by local or circulating norepinephrine and epinephrine. For central pathways, the adrenergic excitation would be equivalent to that produced by noxious events and would consequently evoke pain. In support, evidence is cited for a form of denervation supersensitivity in causalgia and for increased expression of human α-adrenergic receptors after loss of sympathetic activity.