The Role of Gingival Fibroblasts in the Pathogenesis of Periodontitis.

Gingival fibroblasts (GFs) are essential components of the periodontium, which are responsible for the maintenance of tissue structure and integrity. However, the physiological role of GFs is not restricted to the production and remodeling of the extracellular matrix. GFs also act as sentinel cells that modulate the immune response to oral pathogens invading the gingival tissue. As an important "nonclassical" component of the innate immune system, GFs respond to bacteria and damage-related signals by producing cytokines, chemokines, and other inflammatory mediators. Although the activation of GFs supports the elimination of invading bacteria and the resolution of inflammation, their uncontrolled or excessive activation may promote inflammation and bone destruction. This occurs in periodontitis, a chronic inflammatory disease of the periodontium initiated and sustained by dysbiosis. In the inflamed gingival tissue, GFs acquire imprinted proinflammatory phenotypes that promote the growth of inflammophilic pathogens, stimulate osteoclastogenesis, and contribute to the chronicity of inflammation. In this review, we discuss the biological functions of GFs in healthy and inflamed gingival tissue, highlighting recent studies that provide insight into their role in the pathogenesis of periodontal diseases. We also draw parallels with the recently discovered fibroblast populations identified in other tissues and their roles in health and disease. This knowledge should be used in future studies to discover more about the role of GFs in periodontal diseases, especially chronic periodontitis, and to identify therapeutic strategies targeting their pathological interactions with oral pathogens and the immune system.

HDAC3 Regulates Gingival Fibroblast Inflammatory Responses in Periodontitis.

Histone deacetylases (HDACs) are important regulators of gene expression that are aberrantly regulated in several inflammatory and infectious diseases. HDAC inhibitors (HDACi) suppress inflammatory activation of various cell types through epigenetic and non-epigenetic mechanisms, and ameliorate pathology in a mouse model of periodontitis. Activation of gingival fibroblasts (GFs) significantly contributes to the development of periodontitis and the anaerobic bacterium Porphyromonas gingivalis plays a key role in driving chronic inflammation. Here, we analyzed the role of HDACs in inflammatory responses of GFs. Pan-HDACi suberoylanilide hydroxamic acid (SAHA) and/or ITF2357 (givinostat) significantly reduced TNFα- and P. gingivalis-inducible expression and/or production of a cluster of inflammatory mediators in healthy donor GFs (IL1B, CCL2, CCL5, CXCL10, COX2, and MMP3) without affecting cell viability. Selective inhibition of HDAC3/6, but not specific HDAC1, HDAC6, or HDAC8 inhibition, reproduced the suppressive effects of pan-HDACi on the inflammatory gene expression profile induced by TNFα and P. gingivalis, suggesting a critical role for HDAC3 in GF inflammatory activation. Consistently, silencing of HDAC3 expression with siRNA largely recapitulated the effects of HDAC3/6i on mRNA levels of inflammatory mediators in P. gingivalis-infected GFs. In contrast, P. gingivalis internalization and intracellular survival in GFs remained unaffected by HDACi. Activation of mitogen-activated protein kinases and NFκB signaling was unaffected by global or HDAC3/6-selective HDACi, and new protein synthesis was not required for gene suppression by HDACi. Finally, pan-HDACi and HDAC3/6i suppressed P. gingivalis-induced expression of IL1B, CCL2, CCL5, CXCL10, MMP1, and MMP3 in GFs from patients with periodontitis. Our results identify HDAC3 as an important regulator of inflammatory gene expression in GFs and suggest that therapeutic targeting of HDAC activity, in particular HDAC3, may be clinically beneficial in suppressing inflammation in periodontal disease.

Reversal of TREM-1 ectodomain shedding and improved bacterial clearance by intranasal metalloproteinase inhibitors.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is expressed on neutrophils and monocyte/macrophages and amplifies Toll-like receptor-mediated inflammation during infection. TREM-1 also exists in an antagonistic soluble form (sTREM-1) that has been used as a peripheral biomarker in sepsis, though the mechanisms of its release are not entirely clear. The requirement of TREM-1 in single microbial infections is controversial, with some studies showing a protective role and others a contribution to immunopathology. Furthermore, the role of membrane-bound and sTREM-1 in polygenic infections is currently unknown. In a mouse co-infection model where preceding viral infection greatly enhances bacteria co-infection, we now determine a mechanisms for the striking increase in sTREM-1 and the loss of TREM-1 on surface of neutrophils. We identified a matrix metalloproteinase (MMP)-9 cleavage site in TREM-1 and that the increase of MMP-9 in bronchoalveolar lavage fluid mirrors sTREM-1 release. In vitro studies with neutrophils and MMP-9 and the reduction of sTREM-1 in vivo after MMP-9 inhibition verifies that this enzyme cleaves TREM-1. Intriguingly, MMP-9 inhibition significantly reduces bacterial load and ensuing immunopathology in a co-infection model. This highlights MMP-9 inhibition as a potential therapeutic via blocking cleavage of TREM-1.

78,XX testicular DSD syndrome in a mongrel dog.

Because of an apparent sexual ambiguity (enlarged clitoris), a 1-year-old mongrel dog was presented to the clinic. A positive result on a GnRH stimulation test revealed the existence of functional testicular tissue. A midline laparotomy was performed, and gonads resembling testes were resected along with the adherent parts of the uterine horns. Microscopic examination confirmed that the sampled gonads were testes. Cross-sections of the head and tail of the epididymis revealed their typical structures. All layers of the uterine wall were well-developed. The lumen was stellar, covered by columnar cylindrical epithelium, although locally some epithelial cells had changed in height from columnar to flat. The uterine glands were distributed in functional layer of endometrium in a non-uniform way. Cytogenetic analysis based on the evaluation of metaphase plates of blood lymphocytes showed a female karyotype, 78,XX. PCR amplification of the SRY gene was negative in the studied mongrel dog. This canine disorder may be genetically heterogeneous, potentially with a different mutation in different breeds. An autosomal recessive inheritance for the XX male is suggested in such cases. The present case of sex reversal syndrome concerns a non-purebred dog. In mongrels, it is definitely less likely for the defect to be inherited because of a recessive disorder. According to the recently proposed nomenclature, the described case should be classified as 78,XX testicular DSD syndrome.

Parthenogenetic activation of domestic cat oocytes using ethanol, calcium ionophore, cycloheximide and a magnetic field.

The objective of this study was to evaluate parthenogenetic activation of domestic cat oocytes after being exposed to either ethanol, magnetic field, calcium ionophore A23187, or cycloheximide and a combination of these agents. We also wished to evaluate the usefulness of the magnetic field for oocyte activation. In vitro matured oocytes subjected to artificial activation were randomly assigned into eight groups according to activating agents: (1) 10% ethanol; (2) the magnetic field (slow-changing, homogenous magnetic field with low values of induction); (3) 10% ethanol plus magnetic field; (4) 10 microM calcium ionophore A23187; (5) 10 microM calcium ionophore A23187 plus magnetic field; (6) 10% ethanol and 10 microg/mL of cycloheximide; (7) 10% ethanol and 10 microg/mL of cycloheximide plus magnetic field; (8) oocytes were not exposed to any of the activating agents. After activation oocytes were stained with Hoechst 33258 and parthenogenetic activation was defined as oocytes containing pronuclei and second polar bodies or two to four or six nuclei (embryonic cleavage). The total activation rate by using different activation treatments was 40%. The addition of the magnetic field to ethanol or calcium ionophore treatments resulted in increased parthenogenetic activation rates from 47% to 75%, and from 19% to 48%, respectively (P<0.001). Instead, when the magnetic field was added to ethanol and cycloheximide treatment, activation rate decreased from 48% to 30%. Oocytes activated with magnetic field only gave the lowest activation rate (12%). We concluded that a magnetic field can be used as an activating agent, and the combination of ethanol and magnetic field is an effective method for domestic cat oocyte activation.

Application of PCR in situ method for detection of human cytomegalovirus (HCMV) DNA.

Present HCMV diagnosis is relatively slow and inefficient. We applied PCR in situ to 15 samples of human salivary glands, 20 cytospins from blood, and 10 human fibroblast samples in order to detect the presence of HCMV DNA. The results indicate that PCR in situ is an effective and very sensitive method for detection of HCMV infections in variety of specimens.