牙本质涎磷蛋白在小鼠鼻软骨表达的原位杂交研究

目的 :目的 :检测小鼠发育过程中牙本质涎磷蛋白 (dentinsialophosphoprotein ,DSPP)在鼻软骨的表达状况。方法 :采用原位杂交技术 ,观测Balb/C小鼠出生后 1、3、6、8d鼻软骨中DSPPmRNA的表达。结果 :DSPP在 1-6d小鼠的鼻软骨中均有表达。其中 ,3d时表达最强 ,8d时转为阴性。结论 :DSPP在小鼠鼻软骨中的表达具有时空特异性。     

Metabolic mode peculiar to Meckel's cartilage: immunohistochemical comparisons of hypoxia-inducible factor-1alpha and glucose transporters in developing endochondral bones in mice

The middle portion of Meckel's cartilage resembles endochondral bone formation accompanied by chondrocyte hypertrophy and death, cartilaginous matrix calcification, and chondroclastic resorption. We examined Meckel's cartilage specimens from mice mandibles taken on embryonic days 14–16 (E14–E16) using immunohistochemistry for hypoxia-inducible factor-1α (HIF-1α), glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), and glucose transporter 5 (GLUT5), and using enzyme histochemistry for glucose-6-phosphate isomerase (GPI), lactate dehydrogenase (LDH), and cytochrome oxidase (COX), along with the periodic acid-Schiff (PAS) reaction, and compared the results with those of endochondral bones from E16 hind limbs. Periodic acid-Schiff-positive glycogen, HIF-1α, and GLUT immunoreactivity, and GPI, LDH, and COX activities were observed in Meckel's cartilage in E14 and E15 mandibles. In E16 mandibles, hypertrophic chondrocytes showed a transitory loss of HIF-1α immunoreactivity and consumed glycogen, while those closest to the resorption front showed intense immunoreactivity for HIF-1, GLUT3, and GLUT5. Hypertrophic chondrocytes of metatarsals possessed HIF-1α immunoreactivity in the nuclei and diminished COX activity, whereas developing tibias showed weak HIF-1α immunoreactivity even in hypoxic regions characterized by little or no COX activity. These findings suggest that HIF-1α becomes stabilized independently of the concentration of oxygen, and largely contributes to the development and resorption of Meckel's cartilage, probably through shifting the predominant metabolic mode from aerobic to anaerobic glycolysis.     

牙周炎牙龈组织基质金属蛋白酶活性及蛋白表达的研究

目的：探讨来源于宿主的基质金属蛋白酶（MMPs)在牙周炎发病机制中的作用。方法：利用明胶酶活性分析（zymography)和免疫组化方法，检测15例牙周炎患者牙龈组织中和4例健康牙龈中MMP－2、MMP－9的酶活性及蛋白表达。结果：牙周炎牙龈组织以及正常的牙龈组织中均能检测到MMP－2、MMP－9的前体形式（pro-MMP-2和pro-MMP-9)，只有在牙周炎牙龈组织中发现活化形式的MMP－2，没有见到活化形式的MMP－9。免疫组化结果显示：15例成人牙周炎牙龈组织中MMP－2在炎性结缔组织中较正常牙龈阳性表达，而且经图像分析较正常牙龈组织MMP－2阳性细胞表达显著增强（P＜0．05）。结论：提示基质金属蛋白酶MMP－2参与牙周组织破坏过程，在细胞外基质的降解、牙周组织破坏过程中起重要作用。     

Substance P influenced gelatinolytic activity via reactive oxygen species in human pulp cells

Aim To investigate the effects of substance P (SP) on gelatinolytic activity of matrix metalloproteinases (MMPs) in human pulp cells. Methodology Human dental pulp cells were isolated and cultured. Subconfluent cells, between the third and sixth passages, were maintained under serum deprivation for 18 h followed by the treatment of varying doses of SP (1 pmol L^?1, 100 pmol L^?1, 10 nmol L^?1, 1 μmol L^?1 and 100 μmol L^?1). Conditioned media were then subjected to gelatin zymography using 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis minigels containing 1.5 g L^?1 gelatin. The effect of SP on intracellular reactive oxygen species (ROS) was also examined by confocal microscopy. ROS scavenger N‐Acetyl‐l ‐cysteine (NAC, 5 mmol L^?1) was utilized to evaluate the roles of ROS pathway in mediating the impact of SP on cellular gelatinolytic activity. Data were analysed using analysis of variance with Bonferroni correction for multiple comparisons or an unpaired Student’s t‐test. Results Substance P, at levels above 1 μmol L^?1, remarkably enhanced MMP‐2 activity reflected by the band migrating at 66 kDa (P < 0.05). A gelatinolytic band at approximately 44 kDa appeared to be intensified in a SP dose‐dependent manner. In addition, it was demonstrated that SP could induce ROS production in pulp cells and ROS scavenger NAC was further found to significantly reduce MMP‐2 activity (P < 0.05), as well as other bands of gelatinolytic proteinases. Conclusion Substance P can influence gelatinolytic activity in human pulp cells via ROS pathway.     

Immunohistochemical localization of parathyroid hormone-related protein in developing mouse Meckel''s cartilage and mandible

In order to clarify the role of parathyroid hormone-related protein (PTHrP) during Meckel's cartilage and mandibular development, an immunohistochemical study of PTHrP and its receptor, PTH/PTHrP receptor, was designed to examine their localization in the anterior region of Meckel's cartilage including the rostrum, which is known to contribute to the development of the mandible. Meckel's cartilage was first observed on day 13 of gestation and PTHrP was faintly localized in the chondrocytes. On day 16 of gestation, at the stage of elongation and initiation of endochondral ossificastitial in Meckel's cartilage, PTHrP was localized in the chondrocytes located in the area showing interstitial growth and in and around the nuclei of hypertrophic chondrocytes undergoing endochondral ossification. At day 18 of gestation, endochondral ossification was spread over the entire area proximal to the molar region in Meckel's cartilage, except in the mesial fusion site formed by immature chondrocytes. PTHrP was localized in the osteoblasts adjacent to the calcified matrix, but had disappeared from the chondrocytes forming Meckel's cartilage. The localization of PTH/PTHrP receptor was similar to that of PTHrP. These results show that localization of PTHrP is spatially and temporally related to the growth of Meckel's cartilage.     

Long-term nanomechanical properties and gelatinolytic activity of titanium tetrafluoride-treated adhesive dentin interface

ObjectiveThis study investigated the effects of dentin pretreatment with 2.5% titanium tetrafluoride (TiF₄) on nanomechanical properties, and the in situ gelatinolytic activity of the dentin–resin interface, for up to 6 months.MethodsTwenty-four human teeth were prepared by exposing occlusal flat dentin surfaces, and were randomly assigned to experimental groups, according to application or non-application of a TiF₄ pretreatment, and to the adhesive systems (Clearfil SE Bond or Scotchbond Universal). Resin composite (Filtek Supreme Ultra) was built up incrementally on the teeth in all the groups. Then, the specimens were sectioned and randomly selected for evaluation at 24 h, 3 months and 6 months of storage time. The reduced modulus of elasticity (E_r) and the nanohardness of the underlying dentin, as well as the hybrid layer and the adhesive layer were measured using a nanoindenter. Gelatinolytic activity at the dentin–resin interfaces was assessed by in situ zymography using quenched fluorescein-conjugated gelatin at 24 h and 6 months. Statistical analyses were performed with ANOVA and Tukey’s tests.ResultsThere were no differences in E_r and nanohardness values between adhesives systems and pretreatment (p = 0.1250). In situ zymography showed significantly higher gelatinolytic activity after 6 months for all the experimental groups (p = 0.0004), but no differences between the adhesive systems (p = 0.7708) and the surface pretreatment (p = 0.4877). Significance: Dentin pretreatment with 2.5% TiF₄ followed by self-etching adhesive systems did not influence nanomechanical properties or gelatinolytic activity of the adhesive–dentin interface layers, over time.     

Activation of gelatinolytic/collagenolytic activity in dentin by self-etching adhesives

Mild acids are known to activate dentin matrix metalloproteinase (MMPs). All self-etching dental adhesives are acidic (pH 1.5-2.7) and may activate dentin MMPs. The purpose of this study was to compare the ability of several all-in-one adhesives to activate gelatinolytic and collagenolytic activities in powdered mineralized dentin. Powdered dentin made from human teeth was mixed with all-in-one adhesives (Clearfil Tri-S Bond, G-Bond, Adper Prompt L-Pop) or a self-etching primer (Clearfil SE Bond primer) for varying times and then the reaction was stopped by extracting the adhesives using acetone. Fresh untreated mineralized dentin powder had a gelatinolytic activity of 3.31 +/- 0.39 relative fluorescent units (RFU) per mg dry weight (24 h) that increased, over storage time, to 87.5 RFU mg(-1) (24 h) after 6-8 wk. When fresh powder was treated with acidic Tri-S Bond, the gelatinolytic activity increased from 3.24 +/- 0.70 RFU mg(-1) to > 112.5 RFU mg(-1) (24 h) after 20 min and then remained unchanged. Monomers with lower pH values produced less activity. There was a significant, direct correlation between gelatinolytic activity and pH, with Tri-S giving the highest activity. Coating dentin powder with Tri-S resin prevented fluorescent substrates from gaining access to the enzyme, even though it activated the enzyme. In conclusion, self-etch adhesives may activate latent MMP and increase the activity to near-maximum levels and contribute to the degradation of resin-dentin bonds over time.     

In situ detection of matrix metalloproteinase-9 (MMP-9) in gingival epithelium in human periodontal disease

BACKGROUND AND OBJECTIVE: As the periodontal lesion develops, the junctional epithelium migrates apically in conjunction with the dissolution of the most coronal Sharpey's fibers. Because matrix metalloproteinase-9 (MMP-9) has been identified in migrating epithelial cells and invading tumors, we propose that this enzyme is produced by gingival keratinocytes in advanced periodontal lesions. METHODS: To test this idea, biopsies of inflamed gingival tissues were obtained from patients with advanced periodontitis. Healthy gingival tissue samples were utilized as controls. The presence and activity of MMP-9 was evaluated by combining indirect immunofluorescence of gingival tissue samples and gelatin zymography of gingival epithelium separated from connective tissue. RESULTS AND CONCLUSIONS: The staining pattern showed the presence of MMP-9 in junctional and pocket gingival epithelial cells, polymorphonuclear neutrophils (PMNs) and as a scattered deposit along connective tissues of periodontitis-affected gingival tissues. Gelatin zymography permitted the identification of pro-MMP-9 in surcular/pocket epithelium derived from inflamed gingival tissues. Lower levels of MMP-9 were detected in epithelium not exposed to inflammation. These observations suggest a role for MMP-9 in gingival epithelial response to periodontal infection.     

In situ localization of cell synthesis and proliferation in periodontitis gingiva and tonsillar tissue

OBJECTIVE: Previous work indicates that large numbers of B and T cells accumulate in the periodontal soft tissues although we know little about cellular synthetic activity and proliferation at this site. The aim of this study was to examine lymphocytic cell synthetic activity and proliferation in periodontitis gingiva and compare this to a known site of leucocyte proliferation, namely the oro-pharyngeal tonsils. MATERIALS AND METHODS: Messenger RNA (mRNA) and 285 ribosomal RNA (285 rRNA) expressing cells in formalin-fixed/paraffin-embedded gingival and tonsillar tissue sections were detected by in situ hybridisation (ISH) using poly-deoxyribothymidine and 28S probes respectively. In addition S-phase proliferating and cycling cells were also detected by ISH with histone probes and by Ki-67 immunohistochemistry. Ten gingival biopsy samples were obtained from adult periodontitis patients and five tonsillar biopsies from tonsillectomy patients. RESULTS: Both mRNA and 28S rRNA-expressing cells were detected in all the samples tested. Plasma cells showed the strongest signal for the two probes and slight to moderate staining could be seen in epithelium, fibroblasts and endothelial cells. In contrast, gingival lymphocytes were either weakly stained or were unstained for these probes of synthetic activity. In tonsils, most lymphocytes in germinal centres showed moderate staining and mantol zone cells were much more weakly stained. In gingival samples, histone mRNA-expressing and cycling (Ki-67) cells were detected in 4/10, 10/10 cases respectively. These positive cells were mainly basal and suprabasal epithelial cells and a few mononuclear cells, whereas most germinal centre lymphocytes (B cells) were positive for this probe. The number of Ki67 positive cells was greater than histone mRNA bearing cells both in gingiva and tonsillar tissue. In contrast, mantol zone cells (mainly T cells) were sparsely stained by probes of cell proliferation. CONCLUSION: These results indicate that local proliferation of B cells does not occur in periodontitis gingiva in contrast with tonsillar tissue, although plasma cells showed strong synthetic activity in both tissues. T cells did not appear to proliferate greatly nor undergo active synthesis in either of these tissues. These findings substantiate previous hypotheses that specific leucocytes predominate in the gingival tissue through selective homing rather than by local proliferation.     

Examination of the signal transduction pathways leading to activation of gelatinolytic activity by interleukin-1alpha and Porphyromonas gingivalis in human osteosarcoma cells

BACKGROUND: Recently, evidence show that matrix metalloproteinases (MMP) play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of MMPs in human osteosarcoma cells are not fully understood. OBJECTIVES: The purpose of this study was to investigate the gelatinolytic activity in human osteosarcoma cells stimulated with interleukin-1alpha (IL-1alpha) or Porphyromonas gingivalis in the absence or presence of SB203580 (p38 inhibitor), U0126 [mitogen-activated protein kinase kinase (MEK) inhibitor], and LY294002 [phosphatidylinositaol 3-kinase (PI3K) inhibitor]. METHODS: IL-1alpha and the supernatants of P. gingivalis were used to evaluate gelatinolytic activity in human osteosarcoma cells using gelatin zymography. Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the gelatinolytic activity. Results: Gelatin zymography demonstrated that the latent proforms of gelatinases MMP-2 and MMP-9 were released by human osteosarcoma cells. Secretion of MMP-9 was time-dependent by stimulating with IL-1alpha or P. gingivalis. In addition, SB203580, U0126, and LY294002 significantly reduced the IL-1alpha or P. gingivalis-stimulated MMP-9 production, respectively (p < 0.05). However, none of the kinase inhibitors affected the MMP-2 level compared with the control during the 4-day culture period (p > 0.05). CONCLUSIONS: Our findings demonstrated that IL-1alpha and P. gingivalis enhance MMP-9 production in human osteosarcoma cells, and the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of MMP-9. SB203580, U0126, and LY294002 suppress MMP-9 production and/or activity and may therefore be valuable therapeutics in MMP-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis.     

Bone morphogenetic protein 3 expression pattern in rat condylar cartilage, femoral cartilage and mandibular fracture callus

Mandibular condylar cartilage differs from primary cartilage in morphological organization of the chondrocytes and in responses to biomechanical stress and humoral factors. For the first time, we describe the expression of Bmp3 mRNA in relation to types I, II and X collagen mRNA (as determined by in situ hybridization) in chondrocytes of the rat mandibular condylar cartilage, femoral articular cartilage, femoral growth plate cartilage, and temporal cartilage, which transiently appeared in the reparative response stage of mandibular ramus fracture healing. In all cartilages evaluated, Bmp3 was expressed in proliferating chondrocytes that expressed type I collagen in condylar cartilage, articular cartilage, and temporal cartilage appearing during fracture healing. Bmp3 was also found in hypertrophic chondrocytes that expressed type X collagen mRNA in all cartilages evaluated. Furthermore, in remodeling bone, Bmp3 mRNA was strongly expressed in active osteoblast cells in periosteal reaction layers formed after fracture. These findings suggest that Bmp3 expression in a special layer of typical articular cartilage may be regulated by mechanical stress stimulation. We also found that Bmp3 was expressed in the periosteal layers of the bone segments near the fracture site during fracture healing.     

��ͻ���������������ڷ����в����о���չ

髁突软骨和生长板软骨是不同部位的两种软骨,其发育过程均为软骨内成骨。下颌髁突软骨是继发性软骨,由纤维软骨构成;生长板软骨为原发性软骨,由透明软骨构成。二者行使的生理功能不同,在胚胎发生、生长特性、组织结构、软骨细胞的终末方式及对生长因子的反应等方面存在差异。本文就此差异对比做一综述。     

Telomerase activity and in situ telomerase RNA expression in oral carcinogenesis

To investigate the role of telomerase in oral carcinogenesis, we assayed telomerase activity in various oral tissues by a modified telomeric repeat amplification protocol (TRAP) analysis. Also, using digoxigenin-labeled probes, we measured the in situ expression of human telomerase RNA component (hTR) in paired oral squamous cell carcinomas (OSCC) and adjacent non-cancerous matched tissue (NCMT). We detected telomerase activity in three OSCC cell lines, but not in primary oral keratinocytes. In patient samples, most OSCC (36/42, 86%) and oral premalignant lesions (8/12, 67%) possessed telomerase activity. In addition, 6 of 27 (22%) NCMT contained weak telomerase activity. In situ hybridization showed that hTR was expressed in almost all OSCC (23/27, 85%) as well as in the majority of NCMT (20/25, 80%). In most cases, accumulation of hTR was observed both in the nucleus and cytoplasm of epithelial cells. A correlation between hTR expression and more advanced tumor grade was observed. The appearance of telomerase activation and hTR expression during oral carcinogenesis was different. This study indicates that the activation of telomerase is an early and frequent event in OSCC.     

The localization of matrix metalloproteinase-3 and tenascin in synovial membrane of the temporomandibular joint with internal derangement

OBJECTIVES: The aim of this investigation was to study the immunohistochemical localization of MMP-3 and tenascin in the temporomandibular joint and to compare it with control specimens. MATERIALS AND METHODS: Localizations of matrix metalloproteinase-3 (MMP-3) and tenascin in the temporomandibular joint disc and the synovial membrane in 26 human temporomandibular joint samples (internal derangement of TMJ; n = 16, and control; n = 10) by an immunohistological method with monoclonal antibodies specific to human MMP-3 and tenascin. RESULTS: MMP-3 was not distributed in control specimens while it was observed in the internal derangement cases. MMP-3 showed two staining profiles: (1) diffuse staining was observed within the stroma of severely deformed disc with osteophyte and/or disc displacement (three of 16 specimens); and (2) the localization was specifically detected on the surface of severely hypertrophic synovial membrane (six of 16 specimens). The latter localization pattern resembled that of the tenascin on the surface of the severely hypertrophic synovial membrane. CONCLUSION: Comparative localization of MMP-3 and tenascin revealed intense staining for both in the synovial membrane presenting inflammation, proliferation and hypertrophy. These findings suggest that tissue repair and remodeling in the temporomandibular joint disc and the inflammatory hypertrophic reaction in the synovial membrane might proceed at the same time.     

The temporal expression and localization of extracellular matrix metalloproteinase inducer (EMMPRIN) during the development of perio‐dontitis in an animal model

Liu L, Li C, Cai X, Xiang J, Cao Z, Dong W. The temporal expression and localization of extracellular matrix metalloproteinase inducer (EMMPRIN) during the development of periodontitis in an animal model. J Periodont Res 2010; 45: 541–549. © 2010 John Wiley & Sons A/S Background and Objective: We previously demonstrated extracellular matrix metalloproteinase inducer (EMMPRIN) was associated with the matrix metalloproteinases production of human periodontitis. The aim of this study was to investigate the temporal expression and localization of EMMPRIN during ligature‐induced periodontitis in rats. Material and Methods: Periodontitis was inducd in rats by placing a thread around the cervix of the first mandibular molar. Animals were killed 3, 7, 11, 15 or 21 d after ligation. Mandibles were processed for paraffin sections and stained with hematoxylin and eosin or picrosirius red. The distance from the amelocemental junction to the alveolar crest (ACJ–AC) and the area fraction (Area%) of collagen fibers were measured. EMMPRIN was examined by immunohistochemistry and quantified by positive cell counting. Correlation analyses were then performed. Results: Histologically, alveolar bone was gradually destroyed from day 3 to 11 and then stabilized. Collagen fibers were slightly dissociated on day 3 and extensively broken on day 7. They were reconstructed from day 11 to 21. EMMPRIN was localized predominantly in infiltrating cells and adjacent fibroblasts in interdental gingiva. The number of EMMPRIN‐positive cells increased on day 3, peaked on day 7 and then gradually subsided from day 11 to 21. Statistically, there was a moderate positive correlation regarding the ACJ–AC distance (r = 0.552, p < 0.01) and a strong negative correlation with the Area% of collagen fibers (r = ?0.808, p < 0.01). In gingival epithelium, the immunoreactivity was extremely strong in basal layer cells and sulcular epithelial cells in health. It was greatly enhanced in the inflamed conditions on days 3 and 7. In the interradicular bone, EMMPRIN was localized in the osteoclasts on days 3 and 7, as well as in the osteoblasts from day 11 onwards. Conclusion: The expression and localization of EMMPRIN are temporally varied during the development of periodontitis. In addition, the inflammation‐dependent expression of EMMPRIN might be involved in alveolar bone resorption and collagen breakdown.     

甲状旁腺激素相关蛋白mRNA在髁突软骨与生长板软骨发育早期的表达变化

目的研究甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHrP)mRNA在髁突软骨与生长板软骨发育早期的表达变化,初步探讨PTHrP在软骨内成骨中的作用。方法选用胚胎SD大鼠的髁突软骨与生长板软骨作为研究对象,采用原位杂交染色法检测发育早期两种软骨内PTHrP mRNA表达,以图像分析仪进行图像分析。结果胎龄14～19 d,随着软骨发育的成熟,PTHrP mRNA在生长板软骨和髁突软骨中的表达越来越广泛并呈现一定的时空变化,但PTHrP mRNA在两种软骨中的表达部位存在一定的差异。在髁突软骨中,PTHrP mRNA在增殖层和前肥大层强表达;在生长板软骨中,其主要表达在软骨膜细胞和关节周围的软骨细胞,前肥大层中也有较强的表达,增殖层弱表达或不表达。结论 PTHrP mRNA在生长板软骨和髁突软骨中表达部位不同,但均呈现一定的时空变化,提示PTHrP对不同分化阶段的软骨细胞具有一定的调节作用。     

慢性牙周炎与健康牙龈组织中MMP-1的表达和意义

目的:通过检测基质金属蛋白酶-1(MMP-1)在慢性牙周炎牙龈组织中的表达和分布特征,探讨MMP-1在慢性牙周炎发病中的作用和临床意义。方法:收集8例因非牙周疾病而需拔牙患者健康牙龈及24例慢性牙周炎患者牙龈组织,按健康对照、牙周袋深度≤4mm、4~6mm、>6mm分A、B、C、D四组,利用免疫组化方法检测其中MMP-1的表达。结果:正常牙龈组织上皮及固有层MMP-1弱表达,慢性牙周炎患者牙龈组织MMP-1表达明显增高,两者间有显著性差异(P<0.05)。并且MMP-1表达与牙周袋深度间显著正相关(r=0.623,P<0.01)。结论:MMP-1参与慢性牙周炎病变牙周组织的破坏过程,并且其表达随牙周袋深度加深有增加趋势。     

In situ induced demineralization in irradiated and non-irradiated human dentin

The objective of this study was to evaluate the onset of initial demineralization in irradiated and non-irradiated human dentin. Dentin specimens were prepared from the cervical regions of 48 third molars. Either the lingual or the buccal dentin specimen of each tooth was irradiated fractionally up to 60 Gy (2 Gy/d, 5 d/wk). The remaining dentin sample was not irradiated. Two irradiated and two non-irradiated dentin specimens were inserted into both buccal aspects of each 12 intraoral mandibular appliances. The appliances were worn by 12 persons for 5 wk day and night. One side was brushed daily with a fluoride-free toothpaste. On the other side, plaque was allowed to grow. Individual oral hygiene techniques were performed without any fluorides. During meals, the appliance was stored in 10% sucrose solution. After the in situ period, slabs (150 microm) were ground and studied by means of transversal microradiography and microscopic techniques. Concerning mineral loss and lesion depth, ANOVA revealed significant differences between brushed and non-brushed specimens, whereas no differences between irradiated and non-irradiated dentin lesions were found. It is concluded that (in vitro) irradiated dentine is not more susceptible to caries than non-irradiated, if adequate oral hygiene techniques are implemented.     

Analysis of immunoglobulin-synthesizing cells in human dental periapical lesions by in situ hybridization and immunohistochemistry

Immunoglobulin (Ig) kappa (κ) and lambda (λ) light chain mRNA-expressing cells were investigated by in situ hybridization (ISH) to assess the local humoral immune response in human dental periapical lesions. Twenty-seven biopsy samples (17 periapical granulomas and 10 radicular cysts) were examined. Both types of light chain mRNA-positive cells were detected in formalin-fixed/paraffin-embedded tissue sections in all samples. Plasma cells showed weak to strong cytoplasmic staining with both probes and background staining was negligible. The ISH methodology is specific and sensitive in detecting Ig light chain mRNAs and retains cell morphology well. κ to λ ratios showed moderate variability for both granulomas and cysts (mean=1.66±0.85 SD. 1.47±0.51, respectively). There was no significant difference in light chain distribution between granulomas and cysts. Ig κ and λ light chain proteins were also studied by immunohistochemistry (IHC) but the results were disappointing due to excessive background staining. This study confirms that Ig is locally produced in periapical lesions and that the ISH method localises Ig light chain-containing cells better than IHC. The wide variability in κ/λ ratio may support the concept of non-specific multibacterial infection in these lesions.