Hangeshashinto (TJ-14) prevents radiation-induced mucositis by suppressing cyclooxygenase-2 expression and chemotaxis of inflammatory cells

Purpose. Radiation-induced oral mucositis is the most common side effect of radiotherapy in head and neck cancer; however, effective modalities for its prevention have not been established. In this study, we evaluated the effectiveness of Hangeshashinto (TJ-14), a Japanese herbal medicine, for preventing radiation-induced mucositis and elucidated its effect on inflammatory responses, including inflammatory cell chemotaxis and cyclooxygenase-2 (COX2) expression, in an animal model. Methods. Syrian hamsters, 8–9 weeks old, were enrolled in this study. Animals were irradiated with a single 40 Gy dose to the buccal mucosa. Hamsters freely received a treatment diet mixed with 2% TJ-14 or a normal diet daily. The therapeutic effect was determined based on the visual mucositis score, body weight, and histological examination of infiltrated neutrophils and COX2 expression. Results. TJ-14 significantly reduced the severity of mucositis. The percentage with severe mucositis (score ≥3) was 100% in the untreated group and 16.7% in the TJ-14 group (P < 0.05). There was no difference in body weight change between the groups; however, weight gain in the untreated group tended to be suppressed compared to that in the TJ-14 group during the peak period of mucositis. In addition, TJ-14 inhibited the infiltration of neutrophils and COX2 expression in irradiated mucosa (P < 0.05). Conclusions. TJ-14 reduced the severity of mucositis in an animal model by suppressing the inflammatory response. Because TJ-14 is inexpensive and its safety is established, it is a promising candidate for the standard treatment of radiation-induced mucositis in cancer patients (AU)

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Hangeshashinto (TJ-14) prevents radiation-induced mucositis by suppressing cyclooxygenase-2 expression and chemotaxis of inflammatory cells.

PURPOSE: Radiation-induced oral mucositis is the most common side effect of radiotherapy in head and neck cancer; however, effective modalities for its prevention have not been established. In this study, we evaluated the effectiveness of Hangeshashinto (TJ-14), a Japanese herbal medicine, for preventing radiation-induced mucositis and elucidated its effect on inflammatory responses, including inflammatory cell chemotaxis and cyclooxygenase-2 (COX2) expression, in an animal model. METHODS: Syrian hamsters, 8-9 weeks old, were enrolled in this study. Animals were irradiated with a single 40 Gy dose to the buccal mucosa. Hamsters freely received a treatment diet mixed with 2% TJ-14 or a normal diet daily. The therapeutic effect was determined based on the visual mucositis score, body weight, and histological examination of infiltrated neutrophils and COX2 expression. RESULTS: TJ-14 significantly reduced the severity of mucositis. The percentage with severe mucositis (score ≥3) was 100% in the untreated group and 16.7% in the TJ-14 group (P < 0.05). There was no difference in body weight change between the groups; however, weight gain in the untreated group tended to be suppressed compared to that in the TJ-14 group during the peak period of mucositis. In addition, TJ-14 inhibited the infiltration of neutrophils and COX2 expression in irradiated mucosa (P < 0.05). CONCLUSIONS: TJ-14 reduced the severity of mucositis in an animal model by suppressing the inflammatory response. Because TJ-14 is inexpensive and its safety is established, it is a promising candidate for the standard treatment of radiation-induced mucositis in cancer patients.

Hyaluronic acid pretreatment for Sendai virus-mediated cochlear gene transfer.

Gene therapy with viral vectors is one of the most promising strategies for sensorineural hearing loss. However, safe and effective administration of the viral vector into cochlear tissue is difficult because of the anatomical isolation of the cochlea. We investigated the efficiency and safety of round window membrane (RWM) application of Sendai virus, one of the most promising non-genotoxic vectors, after pretreatment with hyaluronic acid (HA) on the RWM to promote efficient viral translocation into the cochlea. Sendai virus expressing the green fluorescent protein reporter gene was detected throughout cochlear tissues following application combined with HA pretreatment. Quantitative analysis revealed that maximum expression was reached 3 days after treatment. The efficiency of transgene expression was several 100-fold greater with HA pretreatment than that without. Furthermore, unlike the conventional intracochlear delivery methods, this approach did not cause hearing loss. These findings reveal the potential utility of gene therapy with Sendai virus and HA for treatment of sensorineural hearing loss.

Sendai virus-mediated gene transfer of the c-myc suppressor far-upstream element-binding protein-interacting repressor suppresses head and neck cancer.

Far-upstream element-binding protein-interacting repressor (FIR) is a transcription factor that inhibits c-Myc expression and has been shown to have antitumor effects in some malignancies. Here, we evaluated the antitumor effects of FIR using fusion gene-deleted Sendai virus (SeV/ΔF) as a nontransmissible vector against head and neck squamous cell carcinoma (HNSCC). Using in vitro and in vivo xenograft mouse models, we observed efficient expression of green fluorescent protein (GFP) following transduction with the SeV/ΔF vector encoding GFP (GFP-SeV/ΔF) into HNSCC cells. In vitro and in vivo studies revealed that administration of the FIR-encoded SeV/ΔF (FIR-SeV/ΔF) vector exerted significant antitumor effects, suppressed c-Myc expression and induced apoptosis in HNSCC. Additionally, the antitumor effects of FIR or the expression of GFP following administration of the FIR- or GFP-SeV/ΔF vector, respectively, were dependent on the multiplicity of infection or titer. Furthermore, the SeV/ΔF vector itself had no cytotoxic effects. Therefore, the SeV/ΔF vector may be safe and useful for the treatment of HNSCC, allowing for high-titer SeV/ΔF vector administration for anticancer gene therapy. In addition, SeV/ΔF vector-mediated FIR gene therapy demonstrated effective tumor suppression in HNSCC, suggesting that this therapy may have the potential for clinical use as a novel strategy for HNSCC treatment.

Pilot study of Biomarkers for predicting effectiveness of ramosetron in diarrhea-predominant irritable bowel syndrome: expression of S100A10 and polymorphisms of TPH1.

BACKGROUND: Serotonin type 3 receptor (5-HT3 R) antagonists are potentially useful therapeutic agents for diarrhea-predominant irritable bowel syndrome (IBS-D). To identify biomarkers predicting effectiveness of the 5-HT3 R antagonist (ramosetron) in IBS-D. METHODS: Irritable bowel syndrome-D Japanese subjects received 2.5 or 5 µg of ramosetron once daily for 4 weeks. Colonic mucosal S100A and tryptophan hydroxylase (TPH) mRNA expression levels were measured before treatment. Genomic DNA was extracted from blood and polymorphisms of TPH1 and TPH2 were analyzed. KEY RESULTS: Forty-two patients (27 men and 15 women, mean age 42 years) with IBS-D were included for analysis. Improvement of IBS symptoms was seen in 26 (61.9%). Baseline S100A10 (p = 0.02) and TPH1 (p = 0.02) expression were significantly higher in the ramosetron responders than in the non-responders. The frequencies of the TPH1 rs4537731G allele in linkage disequilibrium with the TPH1 rs7130929 T allele (11.5% vs 50%, p = 0.003; OR: 12; 95% CI: 2.1-69) along with TPH1 rs211105 C allele (3.8% vs 43.8%, p = 0.0003; OR: 19; 95% CI: 2.1-181) were significantly lower in the responders than in the non-responders. The mean scores of diarrhea at baseline were significantly higher (5.2 vs 3.7, p = 0.005) in patients with TPH1 rs211105 T/T than those with the G allele. CONCLUSIONS & INFERENCES: TPH1 gene polymorphisms and S100A10 expression, which correlate with 5-HT signaling were associated with ramosetron effectiveness in IBS-D, and may possibly lead to prospective identification of the resistance to treatment.

Identification of serum miRNAs as novel non-invasive biomarkers for detection of high risk for early gastric cancer.

BACKGROUND: Many micro-RNAs (miRNAs) are differentially expressed in Helicobacter pylori-infected gastric mucosa and in gastric cancer tissue and previous reports have suggested the possibility of serum miRNAs as complementary tumour markers. The aim of the study was to investigate serum miRNAs and pepsinogen levels in individuals at high risk for gastric cancer both before and after H. pylori eradication. METHODS: Patients with recent history of endoscopic resection for early gastric cancer and the sex- and age-matched controls were enrolled. Serum was collected from subjects before or after eradication and total RNA was extracted to analyse serum levels of 24 miRNAs. Serum pepsinogen (PG) I and II levels were measured using enzyme-linked immunosorbent assay kits. RESULTS: Using miR-16 as an endogenous control, the relative levels of miR-106 and let-7d before and after H. pylori eradication and miR-21 after eradication were significantly higher in the high-risk group than in the controls. H. pylori eradication significantly decreased miR-106b levels and increased let-7d only in the control group. After eradication, the combination MiR-106b with miR-21 was superior to serum pepsinogen and the most valuable biomarker for the differentiating high-risk group from controls. CONCLUSION: Serum miR-106b and miR-21 may provide a novel and stable marker of increased risk for early gastric cancer after H. pylori eradication.

Therapeutic efficacy of acotiamide in patients with functional dyspepsia based on enhanced postprandial gastric accommodation and emptying: randomized controlled study evaluation by real-time ultrasonography.

BACKGROUND: Improvement in subjective symptoms has been reported in functional dyspepsia (FD) patients administered with acotiamide. Improvement was confirmed in meal-related symptoms, such as postprandial fullness, upper abdominal bloating, and early satiety. We examined the mechanism underlying the effects of acotiamide on gastric accommodation reflex (GAR) and gastroduodenal motility in FD patients. METHODS: Thirty-four FD patients (mean age, 40.4 years) were examined ultrasonographically before and after 14-18 days of acotiamide (100 mg t.i.d.) or placebo administration. To assess GAR, expansion rate in cross-sectional area of the proximal stomach was measured after every 100-mL ingestion, using a straw, of up to 400 mL of a liquid meal (consommé soup, 13.1 kcal; 400 mL) in a supine position. Next, we measured gastric emptying rate (GER), motility index (MI, antral contractions), and reflux index (RI, duodenogastric reflux) to assess gastroduodenal motility. Patients also completed a survey based on the seven-point Likert scale both before and after drug administration. KEY RESULTS: Of the 37 cases, 19 and 18 were administered with acotiamide and placebo A respectively, significant difference was observed in GAR between the acotiamide and placebo groups (21.7%vs 4.4%) after 400 mL ingestion. GER significantly accelerated after treatment in the acotiamide group (P = 0.012), no significant differences were observed in MI and RI between the two groups. Improvement rates were 35.3 and 11.8% for the acotiamide and placebo groups. CONCLUSIONS & INFERENCES: Acotiamide significantly enhances GAR and GER in FD patients. Acotiamide may have therapeutic potential for FD patients.

Characterization of autonomic dysfunction in patients with irritable bowel syndrome using fingertip blood flow.

Fingertip blood flow (FTBF) as measured by laser Doppler flowmetry (LDF) measurement is considered an indicator of sympathetic nerve function. We evaluated autonomic function in patients with irritable bowel syndrome (IBS) by assessing FTBF with both LDF and continuous-wave (cw) Doppler sonography. Firstly, the two methods were compared in 40 healthy volunteers. Next, 59 patients with IBS as well as 118 healthy volunteer controls were studied. In the supine position, FTBF in the right index finger was measured with cw Doppler sonography, whereas FTBF in the left index finger was assessed with LDF. After baseline measurement for at least 5 min, the volunteers received sympathetic stimulation from cold stress applied without notification in the form of an icebag (0 degrees C) upon the left forearm for 1 min. The new cw Doppler sonography method can be used in place of the old LDF method for clinical purposes. FTBF velocity before stimulation (V(pre)) was significantly lower in the IBS group than that in the healthy volunteers (P < 0.01). In addition, the time required for FTBF to return to V(pre) after stimulation was significantly longer in the IBS group than that in the control group. (P = 0.02). Thus, measurement of FTBF with cw Doppler sonography can be useful in the assessment of sympathetic nerve function. The IBS patients showed an abnormal FTBF response suggesting the presence of excess sympathetic activity.

Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury.

To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined functional and histological recovery after glial cell line-derived neurotrophic factor (GDNF) gene transfer in a rat RLN crush model. Adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or human GDNF gene (AxCAhGDNF) was injected into the crush site of the RLN. Neurons in the nucleus ambiguus on the crushed side were labeled with X-gal or GDNF immnohistochemistry after AxCALacZ or AxCAhGDNF injection. Reverse transcription-polymerase chain reaction analysis revealed expression of human GDNF mRNA transcripts in brainstem tissue containing the nucleus ambiguus on the crushed side after AxCAhGDNF injection. Animals injected with AxCAhGDNF displayed significantly improved motor nerve conduction velocity of the RLN and recovery rate of vocal fold movement at 2 and 4 weeks after treatment as compared to controls. AxCAhGDNF-injected animals showed a significantly larger axonal diameter and improved remyelination in crushed RLN as compared to controls. Adenoviral GDNF gene transfer may thus promote laryngeal function recovery after RLN injury. Inoculation of adenoviral vector containing the GDNF gene at the site of damage soon after nerve injury may assist patients with laryngeal paralysis caused by nerve injury during head and neck surgery.

Helicobacter pylori-induced atrophic gastritis progressing to gastric cancer exhibits sonic hedgehog loss and aberrant CDX2 expression.

BACKGROUND: The loss of sonic hedgehog is an early change that occurs in the mucosa prior to neoplastic transformation and correlates with the type of intestinal metaplasia. Aberrant expression of CDX has also been shown to correlate with the development of intestinal metaplasia. AIM: To examine CDX2 expression in the non-cancerous mucosa of patients with gastric cancer and compared it to CDX2 expression in controls with intestinal metaplasia. METHODS: Sixty patients who had undergone endoscopic mucosal resection for early gastric cancer and 60 gender- and age-matched controls were studied. Two specimens each were obtained from the greater and lesser curves of the corpus and from the greater curve of the antrum. Expression of CDX2 and sonic hedgehog were evaluated by immunostaining. RESULTS: Gastric cancer was associated with a higher frequency of incomplete intestinal metaplasia (OR = 8.3; 95%CI, 3.7-18.9, P < 0.001). CDX2 negatively correlated with sonic hedgehog expression, however, multivariate analysis revealed that CDX2 correlated with the intestinal metaplasia scores. Sonic hedgehog indices were lower and CDX2 staining in the corpus lesser curve was higher in the cancer group than in the controls. Sonic hedgehog indices in the corpus decreased and CDX2 indices in both areas increased in patients in the ascending order of those without intestinal metaplasia, those with complete intestinal metaplasia and those with incomplete intestinal metaplasia (P < 0.001). CONCLUSIONS: Loss of sonic hedgehog expression and aberrant expression of CDX2 correlates with the type of intestinal metaplasia and may play a role in carcinogenesis.

The time to eradicate gastric cancer is now.

Worldwide gastric cancer remains one of the most common cancers, killing upwards of one million people each year. While the molecular pathogenesis remains unclear, infection with the bacterium Helicobacter pylori is considered a "necessary but not sufficient" cause, not surprisingly as gastric cancer has long been known to be associated with atrophic gastritis. Eradication of H pylori is expected to virtually eliminate gastric cancer and H pylori associated peptic ulcer within approximately 40 years and thus reduce overall mortality. In the USA, the incidence of gastric cancer in the general population is low, reflecting the change in the pattern of gastritis from atrophic to non-atrophic and in the low and decreasing prevalence of H pylori infection in the middle and upper classes. However, the plan for eradication of this important pathogen must be considered within the context of the prevalence and outcome within specific populations.

Hypoacidity combined with high gastric juice nitrite induced by Helicobacter pylori infection is associated with gastric cancer.

BACKGROUND: In patients with Helicobacter pylori infection, the concentration of nitrite in gastric juice is elevated. The degree of elevation correlates with that of inflammation and H. pylori density. AIM: The aim of this study was to examine hypoacidity and high nitrite levels related to H. pylori infection in patients with gastric cancer. METHODS: We studied 88 patients with more than one history of endoscopic mucosal resection (EMR) for early gastric cancer and 88 age-matched controls. Concentration of nitrite in gastric juice was measured by Griess reaction, and serum pepsinogen levels were measured by RIA. RESULTS: Multiple malignant lesions were found in 20 of the 88 patients. Serum gastrin, gastric juice pH and nitrite levels in patients with gastric cancer were significantly higher and pepsinogen I and pepsinogen I/II significantly lower than in control subjects. Pepsinogen I level and I/II ratio were lower and gastric juice pH was higher in the protruded-type group than in the depressed-type group. Pepsinogen I and pepsinogen I/II were lower and gastric juice pH was higher in multiple than in single cases. CONCLUSIONS: Hypoacidity combined with high gastric juice nitrite induced by H. pylori infection is associated with the intestinal type of gastric cancer, especially protruded lesions.

Helicobacter pylori-positive patients with pruritic skin diseases are at increased risk for gastric cancer.

BACKGROUND: Helicobacter pylori infection has been implicated as a possible cause of extraintestinal disorders such as skin diseases. A number of case reports describe patients with skin diseases, such as prurigo nodularis, that are associated with gastric cancer. AIM: The aim of this study was to examine the prevalence of H. pylori infection and the incidence of gastric cancer in patients with pruritic skin diseases. METHODS: The patients were examined for circulating specific IgG antibodies against H. pylori in sera using ELISA. H. pylori-positive patients who were more than 40 years old underwent endoscopic screening for gastric cancer. RESULTS: We examined 134 patients with pruritic skin diseases, including 55 cases of cutaneous pruritus, 21 cases of prurigo chronica multiforme, 15 cases of nummular dermatitis and 43 cases of chronic urticaria. Early gastric cancer was detected in 2/36 (5.6%) patients with cutaneous pruritus and 3/16 (18.8%) with prurigo chronica multiforme. The prevalence of early gastric cancer was 5.6%, which was much higher than that among patients undergoing general endoscopic screening for gastric cancer. CONCLUSIONS: These results suggest that H. pylori-positive patients with pruritic skin diseases may be at increased risk for development of gastric cancer, and endoscopic screening in such patients is recommended.

Effects of electroacupuncture on gastric myoelectrical activity in healthy humans.

The acupuncture point of the wrists (PC6) and the lower legs (ST36) are common points for the treatment of gastric symptoms. However, it remains unclear whether these two acupoints have different effects on gastric myoelectrical activity. We compared the effect of electroacupuncture (EAP) between PC6 and ST36 on gastric myoelectrical activity using surface electrogastrography (EGG). EAP (1 Hz, for 30 min) was applied at either ST36, or PC6, or both acupoints in eight healthy volunteers. EAP at both PC6 and ST36 did not change the percentage of normal slow waves and tachygastria. While EAP at either PC6 or ST36 did not change period dominant frequency (PDF), EAP at both PC6 and ST36 significantly decreased PDF to 78.1 +/- 8.4% of baselines. EAP at PC6 reduced period dominant power (PDP) to 47.2 +/- 5.3% of baselines, while EAP at ST36 increased PDP to 153.6 +/- 28.3% of baselines. EAP at shoulders (sham acupuncture) did not affect the gastric myoelectrical activity. EAP at either PC6 or ST36 shows an opposite effect on PDP, whereas EAP at both PC6 and ST36 has a synergistic effect on PDF. Understanding site-specific effects of acupuncture may contribute to the selection of appropriate acupoints for treating functional GI disorders.

Helicobacter pylori infection and increased nitrite synthesis in the stomach. Inflammation and atrophy connections.

BACKGROUND: We have previously shown that the nitrite content in the gastric juice of Helicobacter pylori-positive patients is significantly higher than that of H. pylori-negative patients and it decreases after eradication of H. pylori. AIM: To examine the relationship between the nitric oxide synthesis in the gastric lumen and histological findings. METHODS: Eighty-five middle aged Japanese patients were studied. Four specimens, each obtained from the greater and lesser curvature of antrum and gastric body were processed for the determination of histopathological scores using the updated Sydney System. Gastric juice was collected endoscopically to determine the concentration of nitrite using a modified Griess reaction. RESULTS: There was a significant positive correlation between the nitrite and the pH levels (r = 0.81, P < 0.001) and between the pH levels and the histopathological scores in the corpus. The gastric juice pH and concentration of the nitrite increased in patients with histological diagnosis of normal, antral-predominant gastritis, pangastritis and corpus-predominant gastritis in due order. CONCLUSIONS: H. pylori infection effects nitric oxide synthesis in the gastric lumen which is mainly connected with hypoacidity. The gastric juice pH is one of the useful markers for corpus dominant gastritis and probably for high-risk group of gastric cancer.

Association between gastric myoelectrical activity and intraluminal nitric oxide.

BACKGROUND: We have previously shown that the intraluminal concentration of NO in H. pylori-positive patients is significantly reduced compared to that in H. pylori-negative patients. AIM: The aim of this study was to evaluate the effect of H. pylori infection on gastric motor activity in relation to the level of NO and nitrite in the stomach in humans. METHODS: Thirty-two H. pylori-negative and 32 H. pylori-positive patients with dyspepsia were studied. Gastric myoelectrical activity was recorded for 24 h using surface electrogastrography. Intraluminal gas and juice were endoscopically collected from the stomach to determine NO and nitrite and nitrate (NOx) levels using a chemiluminescence system. RESULTS: The percentage of tachygastria in the morning preprandial state was significantly higher (P = 0.005) in H. pylori-positive than -negative patients. In H. pylori-negative patients, there was a significant positive correlation between NO levels and the percentage of bradygastria (r = 0.56, P = 0.001) and a significant negative correlation between NOx levels and the percentage of normal electrical activity (r = - 0.57, P=0.001) in the preprandial state. CONCLUSIONS: Gastric motor activity is associated with NO and NOx levels in the gastric lumen. H. pylori infection may play a role in the pathogenesis of abnormal gastric myoelectrical activity.

Citric acid-enhanced Helicobacter pylori urease activity in vivo is unrelated to gastric emptying.

BACKGROUND: In a previous study, the use of a citric acid test meal produced a rapid dose-dependent increase in urease activity that was significantly greater than that resulting from a pudding meal, ascorbic acid or sodium citrate. The mechanism was hypothesized to be related to the ability of citric acid to delay gastric emptying and possibly to enhance intragastric distribution of the urea. OBJECTIVE: To compare the effects of sodium citrate, two doses of citric acid and a pudding meal on gastric motor function. METHOD: Eleven normal healthy volunteers were investigated using non-invasive techniques to measure gastric emptying and gastric motility. We evaluated gastric emptying using the Meretek 13Ceebiscuit solid phase gastric emptying breath test, which employs a 340-calorie biscuit containing 200 mg of the edible 13C-blue-green alga Spirulina platensis, after the administration of test meals of pudding, 2 g and 4 g of citric acid and 2 g of sodium citrate. Electrogastrograms (Digitrapper EGG) were also recorded for 30 min before and 180 min after the test meal. RESULTS: Gastric emptying, as assessed by the half-time (T1/2), was delayed similarly with the pudding (136.8 +/- 9 min) and with 4 g of citric acid (144.5 +/- 7 min) (P > 0.7). Sodium citrate (108.7 +/- 6 min) and 2 g of citric acid (110.1 +/- 6 min) had similar effects on gastric emptying (P=0.986), and were significantly less effective in delaying gastric emptying (P < 0.01) compared to pudding or 4 g of citric acid. The electrogastrograms remained normal and there were no differences among meals and no relation with the gastric emptying results. CONCLUSIONS: The increased intragastric urea hydrolysis associated with citric acid test meals cannot be attributed to delayed gastric emptying. Changes in the intragastric distribution of urea or a direct effect of citric acid on the bacteria (e.g. via the cytoplasmic protein, UreI) are more likely to be responsible.

Localization of receptor activator of NFkappaB ligand, RANKL, in periodontal tissues during experimental movement of rat molars.

The receptor activator of NFkappaB ligand, RANKL, is one of the key regulatory molecules in osteoclast formation and function. We examined RANKL localization in the periodontal tissues during experimental movement of rat molars. To produce orthodontic force, an elastic band was inserted between the upper first and second molars for 4 days, and the dissected maxillae were subjected to light and electron microscopic immunocytochemical examination for RANKL. Expression of RANKL protein was detected in osteoblasts, osteocytes, fibroblasts, and osteoclasts mostly located in resorption lacunae. In osteoblasts, osteocytes, and fibroblasts, RANKL localization was mainly observed in the cytoplasm, the cisternae of rough endoplasmic reticulum and along plasma membranes. In osteoclasts, RANKL was expressed along the ruffled, border membranes and in the cytoplasm, including the clear zone. These results suggest that during tooth movement, osteoclast differentiation and activation are regulated, at least in part, by RANKL, possibly produced by osteoblasts/stromal cells and osteoclasts themselves in the periodontal tissues.