Gingival Sections Research Articles

Activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 6 by Porphyromonas gingivalis regulates programmed cell death in epithelium

Background/purposeGingival epithelial cells form a physiological barrier against bacterial invasion. Programmed cell death (PCD) regulated by pathogen precognition receptors (PRRs) lead to tissue destruction and is closely related to inflammatory diseases. The purpose of this study was to investigate whether nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 6 (NLRP6) expresses in periodontal epithelium and induces PCD of epithelial cells infected by Porphyromonas gingivalis (P. gingivalis), therefore involves in periodontitis. Material and methodsThe expression of NLRP6 was detected in periodontal epithelium from human gingival sections and HaCaT cells stimulated by P. gingivalis. NLRP6 was over-expressed by adenovirus infection in HaCaT or knocked down by siRNA in P. gingivalis infected HaCaT, and the cell death was observed by transmission electron microscopy and flow cytometry analysis. In addition, qPCR and Western blot were performed to determine the expression of NLRP6 and the pyroptosis excutors, caspase-1 and gasdermin D. Enzyme-linked immunosorbent assay were performed to detect the secretion of IL-1β and IL-18. ResultsNLRP6 was up-regulated in both gingival epithelium of patients with periodontitis and P. gingivalis infected HaCaT. Over-expression of NLRP6 in HaCaT led to caspase-1 dependent pyroptosis. Interestingly, knockdown of NLRP6 with siRNA followed by P. gingivalis stimulation inhibited pyroptosis and induced apoptosis. ConclusionUp-regulation of NLRP6 by P. gingivalis in HaCaT led to pyroptosis, while knocking down NLRP6 inhibited pyroptosis and induced apoptosis, which indicated this PRR may play a crucial role in periodontitis by regulating PCD in periodontal epithelium.

Upregulated Vanins and their potential contribution to periodontitis

BackgroundAlthough Vanins are closely related to neutrophil regulation and response to oxidative stress, and play essential roles in inflammatory diseases with clinical significance, their contribution to periodontitis remains to be determined. This research was designed to assess the expression of Vanins in human gingiva, and to define the relationship between Vanins and periodontitis.MethodsForty-eight patients with periodontitis and forty-two periodontal healthy individuals were enrolled for gingival tissue sample collection. Expression levels of VNN1, VNN2 and VNN3 were evaluated by RT-qPCR and validated in datasets GSE10334 and GSE16134. Western blot and immunohistochemistry identified specific proteins within gingiva. The histopathological changes in gingival sections were investigated using HE staining. Correlations between Vanins and clinical parameters, PD and CAL; between Vanins and inflammation, IL1B; and between Vanins and MPO in periodontitis were investigated by Spearman's correlation analysis respectively. Associations between VNN2 and indicators of neutrophil adherence and migration were further validated in two datasets.ResultsVanins were at higher concentrations in diseased gingival tissues in both RT-qPCR and dataset analysis (p < 0.01). Assessment using western blot and immunohistochemistry presented significant upregulations of VNN1 and VNN2 in periodontitis (p < 0.05). The higher expression levels of Vanins, the larger the observed periodontal parameters PD and CAL (p < 0.05), and IL1B (p < 0.001). Moreover, positive correlations existed between VNN2 and MPO, and between VNN2 and neutrophil-related indicators.ConclusionOur study demonstrated upregulation of Vanins in periodontitis and the potential contribution of VNN2 to periodontitis through neutrophils-related pathological processes.

Characterization of junctional structures in the gingival epithelium as barriers against bacterial invasion.

Adherens junctions (AJs) and tight junctions (TJs) are known to play a crucial role in maintaining the physical barrier function of the epithelium. Here, we aimed to characterize the distribution of AJs and TJs throughout the gingival epithelium and to obtain insights into the physiological importance of these junctional structures. Sections of mouse gingival tissue were examined using transmission electron microscopy (TEM) and bio-high voltage electron microscopy tomography. The gingival sections were stained for E-cadherin and JAM-A as markers of AJs and TJs, respectively, and examined using confocal microscopy and lattice structured illumination microscopy. Bacteria within the gingival epithelium were examined using in situ hybridization. Junctional structures, including desmosomes, AJs, and TJs, were observed throughout the gingival epithelium. The expression levels of E-cadherin were particularly low in the granular/keratinized layers of the oral epithelium (OE), while extremely low JAM-A levels were detected in the granular/keratinized layers of the sulcular epithelium (SE). The three-dimensional rendering of the junctional structures revealed that both AJs and TJs in the gingival epithelium formed discontinuous short bands or patches. Interestingly, strong bacterial signals were observed at the granular/keratinized layers of both SE and OE, but a few bacteria were detected within the junctional epithelium (JE) and the basal/spinous layers of the SE and OE. AJs and TJs form a discontinuous barrier throughout paracellular passage in the gingival epithelium; nevertheless, they seem to play an important role in defending against invading bacteria.

Molecular study to the effect of monosodium glutamate on rat gingiva

Background Monosodium glutamate (MSG) is a flavor enhancer widely used throughout the world. It is the salt of glutamic acid, one of the naturally occurring amino acids. It is produced commercially and has classified as generally recognized as safe. Nevertheless, it has been alleged to cause many ills. Objective The present work aimed to evaluate the genotoxic effects of MSG on the rat gingiva. Materials and methods Thirty adult Wistar rats weighting 150–250 g at the age of 6–8 weeks were used in this study. They were partitioned evenly into three groups, each group of 10 rats. Group 1 was the first group that was maintained as control group. Group 2 was the first experimental group while group 3 was the second experimental group. Rats in groups 2 and 3 were administered MSG at two different doses, 15 and 30 mg/kg body weight respectively for 6 weeks. Each gingival tissue sample was divided evenly to be processed and prepared for routine histological examination and molecular study. Results Using hematoxylin and eosin stain, gingival sections presented variable structural changes in both experimental groups including hyperchromatism, pleomorphism of basal cell layer and cytoplasmic basophilia. Group 2 showed areas of epithelial atrophy while group 3 presented acanthosis and hyperkeratosis. Analysis of DNA extracted from gingival tissue samples using five different primers showed marked decrease in the DNA quality. The genotoxic effect of MSG was more declared in group 3 that exhibited the least numbers of bands created with the tested five primers. Conclusion It can be concluded that MSG is genotoxic agent to gingival tissue that has deleterious effects on DNA. These effects were clearly detected in the molecular study and supported by the routine histological findings of the current work.

Role of nuclear factor kappa-B in phenytoin-induced gingival overgrowth

This study investigates the expression of transcription factor nuclear factor-kappa B (NF-κB) and its relation to various cellular mediators that act in the pathogenesis of phenytoin-induced gingival overgrowth. Eighteen epileptic patients had phenytoin-induced gingival overgrowth (PHT-GO), 20 patients with plaque-induced gingivitis (Gingivitis), and 20 periodontally and systemically healthy individuals (Control) were included in this study. The expression of activated NF-κB subunits (p50 and p65), IL-1β, TNF-α and TGFβ-1 levels were examined in the gingival sections obtained from each participant. The results demonstrated a significantly higher expression of p65 in fibroblasts in PHT-GO group with respect to Gingivitis (P<0.05) and control groups (P<0.01). However, we found no statistically significant differences between PHT-GO and Gingivitis groups according to the immunohistochemical staining in macrophages (P>0.05). Immune-reactive TGFβ-1 levels in the gingival connective tissue cells were statistically higher in PHT-GO group with respect to Gingivitis group(P<0.05). Statistically significant correlations were found between the HI and activated TGFβ-1 and p65 levels in PHT-GO group. The results of this study showed that NF-κB is activated in PHT-related gingival overgrowth. This study may provide a basis for future research into specific NF-κB inhibition for preventing of the side effects of this drug.

Lymphocyte subpopulation in healthy and diseased gingival tissue

In this study, infiltrating lymphocytes subpopulation in gingival sections of healthy, inflamed, and periodontitis lesions was investigated. A set of cluster of differentiation (CD) antigen specific monoclonal/polyclonal antibodies to detect different cell types within the tissues was used. These included anti-CD3 (pan T-cell), anti-CD45RO (memory T-cell), anti-CD20 (B-cell), and kappa light chain (plasma cells). Biopsies of gingival tissue were obtained from 17 patients who had clinically healthy gingiva, from 18 patients with gingivitis, and 17 patients with periodontitis.A significantly greater proportion of T-cells (P < 0.00) was observed in healthy gingival and gingivitis tissue samples compared to periodontitis tissue samples. In addition, a greater proportion of B-cells was observed in periodontitis lesions than in the gingival lesions (P < 0.00). The memory T-cells and the kappa light-chain plasma cells were present in both healthy and diseased tissues, suggestive of previous activation by periodontal pathogenic microorganisms.In conclusion, these differences in the relative proportions of B- and T-cells may reflect a difference in the immunopathology of periodontitis and gingivitis lesions.

Tumor necrosis factor‐α stimulates gingival epithelial cells to release high mobility‐group box 1

High-mobility-group box 1 functions as a late-phase inflammatory mediator. It can be released extracellularly by macrophages and necrotic cells through lipopolysaccharide and tumor necrosis factor-alpha. The objective of this study was to clarify the source of high-mobility-group box 1 in chronic periodontitis tissues and tumor necrosis factor-alpha-stimulated gingival epithelial cells, and subsequently elucidate its inducible inflammatory pathway. Chronic periodontitis and healthy gingival sections were stained for high-mobility-group box 1 by immunohistochemistry and immunofluorescence. The amounts of high-mobility-group box 1 released into the gingival crevicular fluid and supernatants from gingival epithelial cells stimulated by tumor necrosis factor-alpha were examined by western blot. The phosphorylation of mitogen-activated protein kinases (MAPKs) in gingival epithelial cells was also examined. High-mobility-group box 1 was detected in the cytoplasm and nucleus of gingival epithelial cells with periodontitis. Western blotting revealed a significant increase in high-mobility-group box 1 expression in the gingival crevicular fluid from periodontitis patients. High-mobility-group box 1 production in gingival epithelial cells was increased following stimulation with tumor necrosis factor-alpha. The molecular dialogue between tumor necrosis factor-alpha and gingival epithelial cells involved modulation of the activities of p38MAPK, Jun N-terminal kinase and p44/42. Interestingly, only phosphorylation of p38MAPK contributed to more than half of the signaling initiated by tumor necrosis factor-alpha-elicited high-mobility-group box 1 release. High-mobility-group box 1 is continuously released from the gingival epithelial cells modulated by tumor necrosis factor-alpha. These findings imply that high-mobility-group box 1 expression and possibly p38MAPK constitute important features in periodontitis.

Are cytokines linked to collagen breakdown during periodontal disease progression?

Evidence of the role of cytokines produced by resident and inflammatory cells during inflammation is well established. The aim of this study was to quantify in healthy and diseased human gingiva the area fraction (AA%) occupied by collagen fibers and the amount of cytokines such as interleukin (IL)-1beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and epidermal growth factor (EGF) to investigate a possible correlation between such cytokines, collagen degradation, and the gingival index. Gingival tissue specimens from 6 healthy controls (group 1), 6 patients with mild gingival inflammation (group 2), 6 patients with moderate gingival inflammation (group 3), and 6 patients with severe gingival inflammation (group 4) were cultured for 72 hours, and the cytokines present in the culture media were quantified using an enzyme-linked immunosorbent assay (ELISA). Paraffin gingival sections from the 24 subjects were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis. The present study revealed significant differences (P < 0.05) between means of AA% in group 1 (53%), group 2 (41%), group 3 (39.5%), and group 4 (35%) for collagen fibers. Compared to controls, there were significant increases of IL-1beta (groups 3 and 4), IL-6, and TNF-alpha (group 3); a significant decrease of IL-4 (groups 2, 3, and 4) and TGF-beta (groups-2 and, 3); and no change of EGF. The collagen AA% was significantly correlated with the amounts of IL-4 and TGF-beta, and significantly inversely correlated with the amounts of IL-1beta for all 3 inflamed groups and IL-6 and TNF-alpha for groups 2 and 3. The present study showed that EGF was not changed in inflamed gingival tissue and that IL-1beta and IL-4 were particularly and intensively correlated with collagen loss. These 2 cytokines could be markers of clinical severity during active periodontitis.

Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy and diseased human gingiva.

The purpose of this study was to quantify the amount of matrix metalloproteinases such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 expressed by human gingival explants in culture media and the area fraction (AA%) of gingival collagen fibers according to the degree of inflammation, to investigate a possible correlation between these enzymes and collagen loss. Gingival tissue specimens from 6 healthy controls (group 1), 17 patients with mild gingival inflammation (group 2), 10 patients with moderate gingival inflammation (group 3), and 9 patients with severe gingival inflammation (group 4) were placed in organ culture for 3 days. The MMPs and TIMPs in the culture media were quantified using zymography, dot blotting, and Western blotting. Paraffin gingival sections were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis. The AA% occupied by collagen fibers significantly decreased from group 1 (53%) to group 4 (35%). The decrease in collagen fibers was inversely correlated with the significant increase in MMP-1, MMP-9, and MMP-13 (dot blotting analysis), with the increase of the active form of MMP-2, and with the active form and proform of MMP-9 (zymography analysis). The present study showed that metalloproteinases, particularly MMP-2, MMP-9, MMP-1, and MMP-13, are involved in the gingival extracellular matrix degradation during periodontitis.

Effect of nifedipine on the expression of p53 protein in rat gingiva

The aim of the present study was to determine the profile of p53 protein expression in gingival tissues after treatment with nifedipine in rats. Rats were treated daily by gastric intubation with or without DMSO alone or DMSO-dissolved nifedipine at concentrations of 15, 30 or 60 mg/kg body weight for 1, 3 or 6 week(s). Gingival width and height were measured macroscopically. Monoclonal antibodies recognizing both wild-type and mutant p53 protein were applied on paraffin-embedded gingival sections using microwave pretreatment and immunohistochemical methods. The gingival width and height were increased in the animals treated with nifedipine at concentrations of 30 and 60 mg/kg body weight. Increased gingival width and height were already seen in the animals treated with 60 mg of nifedipine for 1 week, whereas treatments with 30 mg of nifedipine resulted in increased gingival width and height after treatment for at least 3 weeks. The expression of p53 protein was elevated in the animals treated with 30 or 60 mg of nifedipine. Treatments with nifedipine at the concentration of 60 mg/kg body weight for 1 week induced the expression of p53 protein in the gingival tissues. Treatment with nifedipine in rats led to the inducement of gingival hyperplasia and increase in the numbers of p53-positive gingival epithelial cells by a dose and frequency dependent mechanism, suggesting that p53 protein may play a crucial role in the regulation of nifedipine-induced gingival hyperplasia.

Cell division, synthetic capacity and apoptosis in periodontal lesions analysed by in situ hybridisation and immunohistochemistry.

In this study, we investigated the synthetic and proliferative activity of infiltrating mononuclear cells in sections of granulation tissue from periodontitis lesions in both adult periodontitis (AP) and early onset periodontitis (EOP) patients. We also investigated the role of apoptosis in the remodelling of the inflamed tissue. We utilised a Ki-67 antigen specific antibody and a histone messenger RNA (mRNA) probe to detect cells undergoing cell division in the sections. Oligonucleotide probes for 28S ribosomal RNA and for the detection of poly A mRNA were utilised to detect cells with synthetic capacity. Apoptosis was determined using terminal transferase labelling of fragmented DNA with Biotin labelled dUTP. Biopsies of granulation tissue were obtained from 9 AP patients, from 10 EOP patients and for comparative purposes, biopsies of gingival tissue from 4 patients with AP. There were no differences regarding the relative proportions of cells with synthetic capacity or in the numbers of dividing cells in the periodontitis tissue sections. However, we observed an increase in the number of dividing cells in the AP granulation tissues compared to the AP gingival sections and that these cells were predominantly fibroblast like in appearance. Apoptotic cells consisted mainly of connective tissue cells; mainly fibroblasts with few if any leukocytes being apoptotic other than polymorphonuclear leukocytes. Only a few cyto-phagocytic macrophages were ever observed in the gingival and granulation tissues. We conclude that the turnover of infiltrating leukocytes in inflamed periodontal tissue is low, that they probably arrive at this site by recruitment from distant lymph nodes, and that neither cell division nor programmed cell death significantly alter the numbers of inflammatory cells. On the other hand, fibroblast apoptosis and cell division occur within the periodontium as these are typical processes in the normal turnover and remodelling of these tissues.

Histopathological and clinical studies of acute necrotizing ulcerative gingivitis and evaluation of treatment

We report the clinical data, light and electron microscopic findings, therapy and clinical course of three cases treated for acute necrotizing ulcerative gingivitis. The following results were obtained. 1. An ulcerous lesion was observed in the gingiva of all three cases, with case I also showing a depressed lesion in the ulcerous region. 2. In all three cases, the gingival sections displayed ulceration and showed fibrin deposition and neutrophil infiltration on the surface of the ulcerous region. Congested blood vessels and neutrophil infiltration also were observed in the connective tissue beneath the ulcerous region. 3. Many microorganisms, fibrin, cell debris, and enlarged intercellular spaces of the epithelium were seen on the surface on the ulcerous region. 4. Localized treatment, mainly plaque control, was found to be effective clinically and histopathologically.

The effect of cyclosporin A (CsA) treatment on gingival tissue of patients with Behçet's disease.

Gingival biopsies were obtained at various intervals for a period of two years from 12 patients suffering from Behçet's disease who were under a clinical trial of cyclosporin A (CsA) treatment. The levels of nondialyzable hydroxyproline (Hypro) were determined in the medium of the cultured tissues. Histologic examinations were also performed every three months. Correlations between the CsA blood levels and the levels of nondialyzable Hypro indicated a reciprocal relationship, especially at blood levels of CsA higher than 600 ng/ml. Histologic examination of gingival sections from CsA-treated patients showed swelling of the epithelial cells, formation of perinuclear clear zones, widening of intercellular gaps and formation of several basal cell layers. In addition, foci of PAS-positive material were found in both the epithelium and stroma. It is assumed that the gingival enlargement observed in the CsA-treated patients was not due to an increase in tissue collagen but rather to an increase in epithelium combined with an accumulation of noncollagenous extracellular matrix material.

Tissue localization of Actinobacillus actinomycetemcomitans in human periodontitis. I. Light, immunofluorescence and electron microscopic studies.

Invasion of periodontal tissues by different bacterial morphotypes has been reported in human periodontitis; however, limited information is available as to prevalence, localization and the bacterial species involved. The present study determined prevalence and gingival localization of Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal lesions of juvenile periodontitis patients. Thirty-five gingival biopsies were obtained from 12 juvenile periodontitis patients at the time of periodontal therapy. One additional control biopsy was obtained from each of two adult periodontally healthy subjects, one adult periodontitis patient and one periodontally healthy monkey (Macaca fosibolius). The biopsies were carefully processed to avoid mechanical introduction of bacteria into the tissues and were examined using light and electron microscopy. Rabbit antisera specific for the three A. actinomycetemcomitans serotypes were used for immunofluorescence microscopic localization of A. actinomycetemcomitans antigens in the gingival sections. Immunofluorescence microscopy showed A. actinomycetemcomitans specific antigens in the gingival tissues of 11 of the 12 juvenile patients examined. None of the control specimens showed evidence of A. actinomycetemcomitans antigens in the gingival connective tissue. One specimen from a periodontally healthy subject and the monkey biopsy, however, showed A. actinomycetemcomitans antigens in bacterial plaque on the surface of the crevicular epithelium. Transmission electron microscopic examination showed microcolonies of small gram-negative rods in the connective tissue, as well as single bacterial cells between collagen fibers and in areas of cell debris. In addition to these extracellular bacterial cells, evidence of bacterial cells was also found within gingival connective tissue phagocytic cells. The data from the present study suggest that the gingival tissue in juvenile periodontitis lesions harbors A. actinomycetemcomitans.

Antiserum to intercellular material and basement membrane produced by immunization with oral epithelial cells

Antisera to non-enzymatically released oral epithelial cells were produced in rabbits. Reactivity with gingival sections, by immunofluorescence, was observed for intercellular (IC) and basement membrane (BM) zones. Both IC and BM reactivity could be blocked with Concanavalin A (500 and 1000 micrograms/ml respectively) and anti-human fibronectin serum. Pretreatment with pemphigus serum blocked IC reactivity only. Pemphigus antigen may reside in the material present between coherent epithelial cells and be absent from enzymatically dispersed cells.

Margin placement of esthetic veneer crowns. Part II: Posterior tooth visibility

The amount of posterior tooth visibility was analyzed in a population of 425 subjects. By scoring photographic data, the percentage of posterior teeth that could have a crown margin placed in their occlusal, middle, and gingival sections without showing were calculated. The results of this study indicate that: 1. For esthetic reasons, posterior margin placement should be handled on an individual basis. 2. The routine placement of either supragingival or subgingival margins for esthetics alone on posterior teeth may be over- or under-treating, respectively, a portion of the population. 3. A substantial percentage of the population studied had posterior gingival sections that were visible. A substantial percentage also had gingival sections that were not visible.

Margin placement of esthetic veneer crowns. Part I: Anterior tooth visibility

The visibility of anterior teeth was studied in a population of 425 subjects. Scoring photographic data gave the percentages of anterior teeth that would not show a crown margin if it were placed in incisal, middle, or gingival sections. The results of this study indicate: their anterior teeth which would not show unless the lips were retracted.

Gingival biopsy in thrombotic thrombocytopenic purpura.

Gingival biopsy has been advocated as a readily available, safe, rapid histologic confirmation of the clinical diagnosis of thrombotic thrombocytopenic purpura. Eighteen gingival biopsies from 16 patients with proven thrombotic thrombocytopenic purpura were reviewed. Seven (39%) showed characteristic histologic changes: [1] subendothelial hyalinelike deposits; [2] intraluminal deposits; [3] lack of inflammatory change in vessels and stroma. To assess specificity, gingival sections from 154 patients with oral pathology only and from 50 unselected autopsies were reviewed: 10% to 20% of biopsies from patients with oral pathology only (primarily inflammation) and three of 50 autopsy specimens showed occasional intraluminal deposits but no subendothelial deposits. In addition, other histologic features permitted them to be distinguished from thrombotic thrombocytopenic purpura. We conclude that gingival biopsy in thrombotic thrombocytopenic purpura, although helful in confirming the diagnosis, is less often positive than has been suggested. Biopsy of grossly inflamed gingiva should be avoided.

A METHOD FOR QUANTITATIVE ESTIMATION OF TISSUE COMPONENTS AND NUMBER OF MAST CELLS IN THE GINGIVA.

ABSTRACTA new, systematic and precise method, used for the quantiative estimation of the principal tissue components (epithelial, connective tissue, vascular and inflammatory) of the gingiva and the number of mast cells per unit area of gingival tissue is described in detail. About 500 gingival sections were studied and more thatn 30,000 mast cells were counted with this method.The material consisted of gingival biopsy specimens removed from 137 patients with either clinically normal or infamed and hyperplastic gingiva.The biopsy technique and the processing of the gingival tissues are described. The sections were stained with Hematoxylin and Eosin, Toluidine Blue, Astrablau and Periodic Acid‐Schiff's method.The instruments used in this study, the micrometer component quantitator and an alternative, the light microscope tissue quantitator are also described. Examples of the results obtained are presented and other considerations related to this method are discussed.

Observations on the use of diazo-I-H-tetrazole and phenylglyoxal in protein histochemistry as applied to human gingiva.

A solution of diazo-1-H-tetrazole, freshly prepared by the diazotization of 5-amino-1-H-tetrazole under conditions to avoid explosion, was adjusted to pH 8.8, diluted (1∶1 or 1∶9) with 0.67 M bicarbonate buffer, pH 8.8, and used immediately as a histochemical reagent for demonstrating histidine, tryptophan, and tyrosine residues in deparaffinized sections of frozen-dried human gingiva, rat abdominal skin, and mouse larynx fixed in modified Newcomer's solution. Diazo-1-H-tetrazole reacted histochemically like other diazonium coupling reagents in common use, except that in sections pretreated with bromoacetic acid at pH 7, diazo-1-H-tetrazole staining was increased, rather than decreased as expected. Pretreatment with bromoacetic acid also increased staining in gingival sections exposed to an acetic anhydride-pyridine mixture and then reacted with diazo-1-H-tetrazole. Similarly, pretreatment with bromoacetic acid increased the intensity of Millon's reaction in gingival sections. Sections of human gingiva or mouse larynx pretreated with diazo-1-H-tetrazole stained less intensely with Biebrich Scarlet used respectively at pH 2.62 and 6.50. In test-tube experiments to check the specificity of diazo-1-H-tetrazole for amino acids, only histidine, tryptophan, and tyrosine gave solutions with colours visually distinguishable from the buffer blank. In similar tests a solution of ribonuclease A gave a colour like that given by histidine and tyrosine. Whereas pyridine failed to yield a colour with undiluted diazo-1-H-tetrazole reagent in test-tube experiments, gingival sections exposed to pyridine for 24 hr stained more intensely with diazo-1-H-tetrazole, but diazo-1-H-tetrazole staining of abdominal skin sections was not altered by prior treatment with pyridine. Phenylglyoxal, used as a 1.5% w/v solution inN-ethylmorpholine-acetate buffer (0.2 M acetate) pH 8, blocked the Sakaguchi reaction in human gingival sections. Pretreatment with phenylglyoxal also led to a reduction in their staining by Biebrich Scarlet at pH 2.62, dinitrofluorobenzene, or diazo-1-H-tetrazole. In addition the dimethylaminobenzaldehyde nitrite reaction for tryptophan was reduced. Phenylglyoxal blockade of arginine residues in gingival sections was labile to 1% acetic acid containing 0.05 M choline chloride after 60 min; but in test-tube experiments extending over 320 min, di(phenylglyoxal)-l-arginine hydrochloride was more stable in this acetic acid-choline solution than in water. It is suggested that after treatment of gingival sections with bromoacetic acid at pH 7.0, additional tyrosine residues become available for reaction with diazo-1-H-tetrazole. Pyridine is thought to remove ‘bound lipid’ from gingival epithelium, thereby exposing protein residues reactive with diazo-1-H-tetrazole. The use of diazo-1-H-tetrazole and phenylglyoxal for characterizing amino acid residues of gingival proteins responsible for anionic dye binding is discussed.