Modulation of bullous pemphigoid antigen gene expression by γ-interferon in cultured keratinocytes

Bullous pemphigoid (BP) is characterized by the production of autoantibodies against BP antigens. gamma-interferon (gamma-IFN), a T-cell lymphokine, is known to enhance the expression of several cell-surface proteins. In this study, keratinocytes were cultured in the presence of gamma-IFN, the expression of BP antigen protein was examined by flow cytometry and BP antigen messenger RNA (mRNA) (encoding 230-kDa protein) was quantified by slot-blot hybridization. The results indicated that BP antigen gene expression by keratinocytes was upregulated by gamma-IFN. This enhancement of gene expression was detected at both the protein and mRNA level, suggesting pretranslational regulation. These results imply the involvement of not only humoral immunity but also cell-mediated immunity in the development of BP.     

Hypochlorous acid induced activation of human neutrophil and gingival crevicular fluid collagenase can be inhibited by ascorbate

Abstract— Interstitial collagenase either obtained from human neutrophils by phorbol myristate acetate (PMA) induced degranulation or isolated from human gingival crevicular fluid was found to be activated by addition of an oxidative agent, hypochlorous acid (HOCl). Collagenase released by PMA stimulated neutrophils was completely in latent form but underwent partial autoactivation during 16 h incubation at 22°C in the presence of soy bean trypsin inhibitor. The partial autoactivation was potentiated to complete activation of released collagenase after addition of exogenous HOCl. Ascorbate prevented this activation of neutrophil collagenase. Isolated human gingival crevicular fluid collagenase represented an apparent M_r of 70 kD in completely latent form, whereas 70/54 kD enzyme species were detected for partially autoactive form of the enzyme. Western blot analysis of gingival crevicular fluid using a polyclonal antibody raised against purified human neutrophil collagenase revealed the same 70/54 kD molecular forms of the enzyme. The latent gingival crevicular fluid collagenase was also activated by HOCl and this activation could be prevented by ascorbate. Activation of the 70 kD latent collagenase by HOCl as well as by other non-proteolytic activators such as an organomercurial compound (phenylmercuric chloride) and a gold (I) compound (gold thioglucose) was not associated with detectable changes in apparent M_r, whereas trypsin activation resulted in fragmentation of 70 kD enzyme to 54 kD species. Our results provide further evidence for the neutrophil origin of gingival crevicular fluid collagenase and suggest that, in addition to proteolytic activation, oxidative and antioxidative agents seem to be able to regulate neutrophil collagenase activity.     

Intracellular replication of fusobacteria requires new actin filament formation of epithelial cells

We examined survival and replication of fusobacteria inside epithelial cells. Subconfluent cultures of HaCaT keratinocytes were infected with five bacterial strains representing three Fusobacterium species: F. nucleatum, F. necrophorum, and F. mortiferum. Adhesion and invasion of the bacteria were assayed before and after antibiotic treatment that killed the adhered and extracellular bacteria. The number of live fusobacteria was examined by bacterial culturing after sonication of the epithelial cells. The role of host cell cytoskeleton functions was examined by treating the epithelial cells with cell function inhibitors. Number of viable epithelial cells was measured with the CellTiter96 kit. The tested Fusobacterium species adhered to and invaded the epithelial cells, and multiplied intracellularly for several hours. Thereafter, the intracellular number of bacteria rapidly declined. Concomitantly, viable fusobacteria were detected in the culture medium. Treatment of the infected epithelial cells with an actin formation inhibitor markedly reduced the number of living intracellular fusobacteria. Newly formed actin filaments were seen by confocal microscopy in the epithelial cells associated with the invaded bacteria. Fusobacteria infection did not reduce the number of viable epithelial cells in culture. Thus, fusobacteria are able to adhere to and invade epithelial cells, and survive under aerobic conditions. This property may enable them to survive in mucosa and participate in various disease processes of oral and pharyngeal tissues.     

Fibronectin fragmentation induced by dental plaque and Bacteroides gingivalis

Abstract – Degradation of fibronectin (FN) by subgingival and supragingival plaque and Bacteroides gingivalis (Bg) was studied in vitro. The degradation of FN by both types of plaque was relatively rapid, continuous but incomplete. Some differences were found between supra-and subgingival samples. Supragingival plaque extracts produced several FN fragments of 110–180 kd during short incubations, of 15–60 min. The predominant fragment after overnight incubation was a 110 kd polypeptide. With subgingival plaque extract a more extensive degradation of FN was noted. The main degradation product was a 120 kd fragment after overnight incubation. Several peptide fragments were released from fibronectin by Bg extracts. Their molecular size was different from those produced by trypsin, elastase or dental plaque. When cell extracts of Bg were fractionated by high performance liquid chromatography, three separate peaks of fibronectin degrading activity were obtained. Two of those peaks also contained trypsin-like enzyme activity. The degradation of fibronectin and the subsequent formation of biologically active peptides may have many effects in periodontal pockets. These may include modifying effects on plaque growth and wound healing.     

Dissolution of bovine pulp tissue by endodontic solutions

Pulpal tissue was incubated at 37°C for 10 min with various solutions used for root canal therapy. The dissolved material was assayed for hydroxyproline (HYP) and total phosphate, and the insoluble residue for HYP and dry weight. Sodium hypochlorite (NaOCl) at 5% and diluted to 2.5% showed the strongest solvent capacity measured as loss of HYP and weight from the tissue. Dilution to 0.5% significantly decreased the effectiveness of NaOCl. The absence of HYP from the NaOCl extracts suggested decomposition of this amino acid. The other solutions tested were far inferior in their ability to dissolve pulpal tissue. The demineralizing solutions tested were poor solubilizers of soft tissue; however, they caused a considerable increase in the amount of phosphate released.     

Demonstration of cellular retinoic acid binding protein in cultured human skin fibroblasts

Previous studies have indicated that retinoids, such as all-trans-retinoic acid and 13-cis-retinoic acid, can modulate connective tissue metabolism in human skin fibroblast cultures. Such effects could be mediated through binding of these retinoids to specific cellular binding proteins. In the present study we have demonstrated cellular retinoic acid binding protein using both whole cell and cytosol binding assays with [3H]all-trans-retinoic acid or [3H]13-cis-retinoic acid as the ligand. Specific binding of [3H]all-trans-retinoic acid could be demonstrated by both techniques and the binding could be displaced by unlabelled all-trans-retinoic acid and 13-cis-retinoic acid, but not by retinol or RO-10-9359 (etretinate) in a 100-fold excess. Gel filtration chromatography of the cytosol proteins after incubation with [3H]all-trans-retinoic acid demonstrated that the specific binding protein had an apparent molecular weight of approximately 15 000 daltons. Thus, the cellular retinoic acid binding protein demonstrated in human skin fibroblasts may mediate the effects of the retinoids on connective tissue metabolism in these cells.     

QUANTITATIVE HISTOCHEMICAL ANALYSIS OF MAST CELLS AND SENSORY NERVES IN PSORIATIC SKIN

To study the elements of neurogenic inflammation in psoriatic skin, morphological contacts were examined between mast cells and sensory nerves containing the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP) or vasoactive intestinal polypeptide (VIP). Because mast cells in psoriatic lesions appear in great numbers at the basement membrane (BM) zone, neuropeptide–mast cell contacts with the BM were also counted. A double stain for active mast cell tryptase and the neuropeptides was applied and the contacts were quantitated morphometrically. Sensory nerve–mast cell contacts were also studied three-dimensionally with a confocal laser scanning microscope. Increases in the contact values of SP and CGRP with mast cells, as well as with the BM, were obtained in developing (1–3 weeks) lesions when compared with their non-lesional controls. This increase reached statistical significance in mature lesions. In contrast, the corresponding contact values for VIP were decreased. By confocal microscopy, a close association between mast cells and sensory nerves was observed in the lesional dermis. Since tryptase is known to degrade CGRP but not SP, neurogenic stimuli, mainly via SP, can result in degranulation of mast cells, which release substances to enhance inflammation. At the BM zone in psoriatic lesions, the numerous mast cells loaded with tryptase can promote degradation of BM components and allow entry of various mediators to interact with keratinocytes.     

A THREE-STEP PROCEDURE FOR ASSESSING BIOEQUIVALENCE IN THE GENERAL MIXED MODEL FRAMEWORK

Bioavailability data arising from a standard two-period cross-over study are routinely analysed to establish bioequivalence between test and reference formulations. Current regulatory guidelines only require evidence of equivalence in average bioavailability for the assessment of bioequivalence. Under normality assumptions, this is achieved by demonstrating equivalence between the formulation means (step 1). However, the equivalence of formulation variances should also be assessed to get evidence of population bioequivalence (step 2), since a difference in variability of bioavailability may also pose significant problems in drug safety and efficacy. On the other hand, even population bioequivalence does not ensure that an individual subject could be expected to respond similarly to the two formulations. Therefore, whenever individual bioequivalence is the ultimate goal, the magnitude of intra-subject correlation should always be examined as the final stage (step 3). In this paper, these three successive concepts of bioequivalence are cast into the general mixed model framework and a stepwise testing procedure for the global assessment of bioequivalence is proposed. In addition to this, important issues addressed in the regulatory guidelines, such as verification of the model assumptions and application of the log-transformation, are discussed. Lastly, an example is presented to illustrate the proposed three-step procedure on the original and log-transformed scale of measurement.