RUSSELL BODIES DEVELOPMENT IN ASSOCIATION WITH...

* Lecturer, Oral Biology Deptartment, Faculty of Oral and Dental Medicine, Cairo University.

INTRODUCTION

Diabetes mellitus is a multi-factorial group of disorders associated with an abnormality in glucose metabolism. It is characterized by metabolic abnormalities and long term complications

involving the eyes, kidneys, nerves, vasculature and periodontium (1). Hyperglycemia associated with diabetes leads directly to activation of the mitogen-activated protein (MAP) kinases or protein kinase C (PKC) pathways, both of which stimulate cytokine

مالحظاتصور 4 لونصور 1 لونص األلوانعدد الصفحاتالترقيمرقم املقالة

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RUSSELL BODIES DEVELOPMENT IN ASSOCIATION WITH APOPTOTIC CHANGES IN DIABETIC HUMAN GINGIVA

Marwa M.S. Abbass * and Rehab A. Abdel Moneim *

ABSTRACT

Background: Periodontal disease is considered the sixth major complication of diabetes mellitus. Apoptosis plays a functional role in limiting diabetic repair. The immune system has been shown to play an important role in the initiation and progression of periodontal disease. Russell bodies, an uncommon finding that may be associated with chronic inflammatory conditions. T cells might promote the formation of Russell bodies through the release of soluble factors, such as IL-2 which acts in a paracrine fashion to support the function of T cells.

Methods: Gingival biopsies were collected from 10 Type 2 diabetic patients with periodontitis (Diabetic group) and from 10 donors with no known history of diabetes mellitus or periodontitis (Control group).The sections were examined histologically, immunohistochemically, histomorphometrically and by immunofluorescence technique.

Results: Histopathological examination of the diabetic gingiva revealed the association of Russell bodies with the numerous inflammatory cells detected in the lamina propria. This was further assured by increased immunoreaction for CD138. Also, there was increased immunoreactivity for caspase-3 and IL-2. The histomorphometric analysis supported the immunohistochemical results as there has been a significant increase in the area percentage of caspase-3, CD-138 and IL-2 immunoreaction in the diabetic gingiva in comparison to the control group. Immunofluorescence labeling revealed increased reactions for IgA and IgM in the diabetic group.

Conclusions: High levels of caspase-3 and IL-2 detected in Type 2 diabetic gingiva results in the formation of Russell bodies (Mott cells) associated with increased levels of IgA and IgM

KEY WORDS: diabetes; gingival; Russell bodies; apoptosis; IL-2; Ig M; IgA

(2) Marwa M.S. Abbass and Rehab A. Abdel MoneimE.D.J. Vol. 60, No. 2

production such as tumor necrosis factor-α (TNF-α) and promote inflammation (2). Advanced glycation end-products that accumulate during prolonged hyperglycemia also promote inflammation and induce oxidative stress (3, 4).

Apoptosis plays an important role in several diabetic complications. These include apoptosis of neuronal cells in diabetic neuropathy (5), myocardial apoptosis, which plays a role in cardiac pathogenesis (6) and apoptosis of mesangial cells that occurs in diabetic nephropathy (7). Furthermore, abnormal apoptosis has been implicated in periodontal diseases (8, 9) and diabetes (10, 11). The molecular events regulating apoptosis are complex and involve genes that are both proapoptotic and antiapoptotic.

The immune system has been shown to play an important role in the initiation and progression of periodontal disease. Cell mediated immunity is reported to play a protective or aggressive role in the pathogenesis of periodontal disease. Altered immune function in diabetic patients with periodontitis have been cited in many immunological studies (12, 13). Furthermore, previous studies showed a significantly elevated serum immunoglobulins and complement factors in diabetic patients with periodontitis (14, 15). Fontana et al., also concluded that a systemic factor might be responsible for promoting the local pathological alterations, which produce gingivitis and periodontitis in diabetes patients. Little research has been carved out to estimate the levels of immunoglobulins in the gingival tissues of diabetic patients with periodontitis (13).

There are five immunoglobulin classes synthesized by B-lymphocytes that are designated IgM, IgG, IgA, IgE and IgD (16). An IgM is the first antibody to appear in the circulation after stimulation of B-lymphocytes. An IgG is the classical gamma globulin and is the major circulating antibody. IgG appears about 24-48 hours after antigenic

stimulation and continues the antigen antibody interaction begun by IgM . On the other hand, an IgA appears selectively in the sero-mucous secretion such as saliva, nasal fluid… etc. It protects the exposed surfaces of the body against the micro-organisms and provides resistance in the respiratory and gastrointestinal tract, possibly by inhibiting the attachment of these parasites to the tissues (17).

Russell bodies, which are eosinophilic, large, homogenous immunoglobulin-containing cytoplasmic inclusions, usually found in plasma cells undergoing excessive synthesis of immunoglobulins (18-20). The reason that these immunoglobulins are not eliminated and crystallize in the cytoplasm remains to be established. Plasma cells filled with Russell bodies, also known as Mott cells, have been associated to immunoproliferative disorders such as plasmocytoma(21-23). The Mott cells can be demonstrated by PAS method and are immunopositive for the antibodies CD79a, CD138, and kappa and lambda immunoglobulin light chain (24-26). T cells may promote the formation of Mott cells through the release of soluble factors, such as interleukin-2 (IL-2) (27).

IL-2 can be used by cells expressing IL-2 receptors (IL-2R) either the intermediate-affinity receptor dimer of IL-2Rb (CD122) or the common gamma chain (CD132), or the high-affinity trimeric receptor comprising IL-2Ra (CD25), IL-2Rb, and gamma chain. The intermediate-affinity receptor is more broadly expressed on T cells, natural killer (NK) cells and monocytes, whereas the high-affinity receptor is only constitutively expressed on regulatory T-cells (Treg) (28).

From the fore mentioned, the present study was designed to determine the adaptive apoptotic and inflammatory alterations accompanying diabetes in human gingiva with the possibility of occurrence of the so-called Russell bodies.

RUSSEL BODIES IN DIABETIC GINGIVA (3)

MATERIALS AND METHODS

Tissue samples

Tissue samples used in this study were obtained from patients of the Faculty of Oral and Dental Medicine, Cairo University, during periodontal surgery, surgical extraction or preprosthetic surgery. All patients gave their written consent to participate in the study. Use of human material in this research was in accordance with the Declaration of Helsinki (29). Only male patients were selected for this study to avoid the possible adverse effects of female ovarian hormones that could increase the gingival inflammation (30). The age ranged from 40 to 60 years. Smokers were excluded from this study.

The gingival biopsies (one per person) were collected from 10 diabetic patients (Type 2 diabetes mellitus) with periodontitis (Diabetic group) and from 10 donors with no known history of diabetes mellitus or periodontitis (Control group). The specimens were collected from the buccal or the lingual free or interdental gingiva of mandibular posterior teeth in case of the diabetic group and from maxillary posterior teeth in the control group.

Periodontal evaluation was performed for all the patients using:

1. Gingival index (31).

2. Periodontal pocket depth (PPD). The site where the biopsy was taken in the diabetic group had a pocket depth of at least 4 mm.

3. Clinical attachment level (CAL).

4. Radiographic examination using panoramic technique. Screening criteria for chronic periodontitis was the presence of at least 5 sites with ≥ 4mm horizontal alveolar bone loss on radiographs.

All patients had a blood glucose test performed prior to biopsy to confirm their non-diabetic or diabetic status. In the diabetic patients, the glycated

hemoglobin (HbA1c) test was used to monitor the patient’s overall glycemic control. The patients with HbA1c ≥ 6.5% were considered diabetics according to the 2010 American Diabetes Association Standards of Medical Care (32).

All the gingival tissue samples were washed in saline solution and fixed in 4% buffered formalin. The specimens were dehydrated in ascending grades of ethyl alcohol, cleared in xylene and embedded in paraffin. Sections of 4-5 μm in thickness were obtained and collected on positively charged microscope slides. Tissue sections were deparaffinized and rehydrated before histological staining and immunolabeling.

Hematoxylin and Eosin (H&E) staining

A set of sections from the two groups was stained with hematoxylin and eosin solutions (Sigma, St. Louis, MO, USA), dehydrated, mounted and examined in the Olympus light microscope equipped with digital camera and image analysis software for histological evaluation of morphological changes.

Immunohistochemistry

Another set of sections were incubated in hot oven for 2 hours at 56°C, deparaffinized in xylene and rehydrated by ethanol series ending with pure H2O (Millipore Corporation, Temecula, CA, USA). After 5-minutes washing in phosphate buffer saline (PBS), sections were incubated in 0.05 mg/ml proteinase K in 0.05 M Tris-HCl, 0.01 M Ethylene diamine tetraacetic acid (EDTA) and 0.01 M NaCl, pH 7.8 for 10 minutes at 37°C. After two washes with PBS, unmasking of the antigens was carried out using antigen retrieval citrate buffer solution for 10 min in boiling water. Then the sections were placed in a humid chamber and the endogenous tissue peroxidase was blocked with 3% hydrogen peroxide for 5 min. Incubation with bovine serum albumin for 20 min was performed to reduce unwanted nonspecific reactions. Without washing, the sections were incubated with the


primary antibodies overnight at 4°C. The primary antibodies used were anti-caspase-3 active form (Millipore Corporation, Temecula, CA, USA), anti- IL-2 (R&D Systems, Minneapolis, MN, USA) and anti- CD 138 (R&D Systems, Minneapolis, MN, USA) with dilutions 1:100. In the next day, after washing in PBS, the sections were incubated with secondary universal antibody (Vectastain Universal Elite ABC- peroxidase kit, Vector Laboratories) and then with the Avidin-Biotin complex (ABC) (Vectastain Universal Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s protocol. The substrate 3,3’-diaminobenzidine (DAB) was applied for the same amount of time on all labeled sections until development of desired brown color. Finally, the sections were counter-stained with Mayer’s hematoxylin (Sigma, St. Louis, MO, USA) for 30 sec. to visualize tissue topography. The negative control was obtained by omitting the primary antibody from the protocol outlined above.

Immunofluorescence

A third set of sections from each group was labeled using direct immunofluorescence technique. The sections were incubated in hot oven for 2 hours at 56°C, deparaffinized in xylene, rehydrated in descending grades of ethanol and afterward were kept in PBS. Microwave treatment with freshly prepared buffer consisting of EDTA pH 9.0 (DAKO) for 20 min has been carried out. Then, the slides were cooled at room temperature and incubated with polyclonal Fluorescein isothiocyanate (FITC)-labelled rabbit antihuman antibodies (DAKO): IgA (dilution 1 : 20) and IgM (dilution 1 : 20). Incubation with antibodies was carried out overnight at 4°C in a moist chamber. Negative controls were carried out with incubation slides with PBS in the absence of the primary antibody and always yielded negative results. All sections were mounted in aqueous medium and examined under a dark field ultraviolet fluorescence microscope (Olympus, BX41). The intensity of immunofluorescence in gingival tissues

was scored semiquantitatively on a scale of 0 (lack of reaction), weak (+1), moderate (+2) and strong (+3).

Histomorphometric analysis

The image analysis was performed using a Leica microscope equipped with a digital camera and software (Leica Qwin 500). The image analyzer was calibrated automatically to convert the measurement units (pixels) produced by the image analysis program into actual micrometer units. In the immunohistochemistry treated sections, the image analysis system was used to assess the area percentage of caspase -3, CD-138 and IL-2 immunoreactivity in the inflammatory cells and the Russell bodies. Ten fields were measured for each specimen. After grey calibration, the image was transformed into a grey delineated image to choose areas exhibiting positive reactivity with accumulation of all grades of reactivity (minimum, maximum and median grey). Areas of positive reaction were then masked by a blue binary color. Mean values were obtained for each specimen. All the calculations were performed in relation to a standard measuring frame of an area 118476.6 μm2.

Statistical analysis

Data obtained from histomorphometric analysis were statistically described in terms of mean ± standard deviation (± SD). Data distribution was tested using Kolmogorov-Smirnov test for each of the two groups. All data were shown not violating the normal assumption (p > 0.05). Accordingly, comparison was done using Student t test for independent samples. A probability value (p value) less than 0.05 was considered statistically significant. All statistical calculations were performed using Microsoft Excel 2007 (Microsoft Corporation, NY, USA) and SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows. Power calculations between the two groups were done using PS Power and Sample Size Calculations Software, version 2.1.30 for MS Windows (William D. Dupont and Walton D. Vanderbilt, USA)


RESULTS

General morphology

In comparison to the healthy gingiva (Fig.1A), the lamina propria in the gingiva of diabetic patients showed strong inflammatory cells infiltration accompanied by numerous Russell bodies. Furthermore, the collagen fibers exhibited signs of degeneration in certain areas. In other areas, hyalinization or even thickening of the fibers was detected (Fig.1B).

Immunohistochemistry

In the diabetic group, a strong immunohisto-chemical reaction for caspase-3, CD-138 and IL-2 was observed in the inflammatory cells. Also, the multiple Russell bodies demonstrated among the inflammatory cells exhibited moderate to intense immunoreactivity along the entire field (Fig. 2B, 2D, 2F). The endothelial cells lining the blood ves-sels revealed strong caspase-3 reaction, while the

smooth muscle cells showed mild immunoreaction (Fig. 2B). On the other hand, in the gingiva of the control group, mild immunohistochemical reaction for caspase-3 and CD-138 as well as negative IL-2 immunoreactivity was revealed among the few de-tected inflammatory cells in addition to the endothe-lial cells (Fig. 2A, 2C, 2E).

Fig. (1) A photomicrograph of the lamina propria from control healthy gingiva (A) and from diabetic gingiva (B). In the diabetic gingiva there has been strong inflammatory cells infiltration (arrows) invaded by multiple Russell bodies (arrowheads) and thick bundles of dense collagen fibers (c) (B). (H & E, 400x).

Fig. (2) A photomicrograph of caspase-3, CD-138 and IL-2 immunolocalization in the gingival tissues from healthy control group (A, C, E) and from diabetic group (B, D, F). In the control group, the few detected inflammatory cells (arrowheads) revealed mild immunoreaction for anti-caspase-3 antibody (A), anti-CD-138 antibody (C) and negative IL-2 immunoreactivity (E). The endothelial cells (thick arrow) revealed mild IL-2 immunoreaction (E). In the diabetic group, strong immunoreaction was detected in the inflammatory cells (arrows) and moderate to intense immunoreactivity in the Russell bodies (arrowheads) for anti-caspase-3 (B), anti-CD-138 (D) and anti-IL-2 antibodies (E). The endothelial cells (thick arrows) revealed strong caspase-3 immunoreaction (B), while the smooth muscle cells (s) exhibited mild reactions for anti-caspase-3 and anti-CD-138 antibodies (B, D). (Peroxidase labeling technique, 400x, insets 630x)


TABLE (1) The area percentage of Caspase -3, CD-138 and IL-2 immunoreactivity in the inflammatory cells and the Russell bodies in the gingival tissue from diabetic and control groups

Caspase-3 % CD-138 Il-2

Diabetic (n=10) 19.99±6.28 11.39±3.384 14.72±5.62

Control (n=10) 1.68±0.7 2.02±0.51 3.10±1.44

p value < 0.001* < 0.001* < 0.001*

Data presented as mean±SD; * statistically significant difference; n – number of subjects

The histomorphometric analysis data supported the immunohistochemical results as there has been a high significant increase in the area percentage of caspase-3, CD-138 and IL-2 immunoreaction in the gingiva of diabetic group in comparison to the control group (Table 1).

Immunofluorescence

There has been a strong IgA immunoreaction in some of the detected inflammatory cells, while moderate IgA reaction has been revealed in the Russell bodies in the lamina propria of the diabetic group (Fig. 3B). Extracellular IgA immunoreactions have been detected in the diabetic group (Fig. 3B). The collagen fibers in the gingiva of the control group almost exhibited weak IgA immunoreactivity (Fig. 3A).

Nearly all the inflammatory cells and Russell bodies in the lamina propria of the diabetic gingiva showed strong IgM immnoreaction (Fig. 3D). Moderate IgM reaction has been detected in the collagen fibers, endothelial cells and smooth muscle cells. No extracellular expression of IgM has been demonstrated in the diabetic group (Fig. 3D). Moreover, the collagen fibers of the control group revealed weak IgM reaction (Fig. 3C).

Fig. (3) A photomicrograph of immunofluorescent labeling for IgA and IgM in the gingiva from healthy control group (A, C) and from diabetic group (B, D). The collagen fibers (c) from the healthy gingiva exhibited almost weak immunoreactivity for both immunoglobulins (A, C). The diabetic group revealed strong immunoreaction for IgA and IgM in the inflammatory cells (arrows) and moderate to strong reaction in the Russell bodies (arrowheads) (B, D). A strong IgA extracellular immunoreactivity has been detected in the diabetic gingiva (B). Moderate IgM immunoreaction has been demonstrated in the collagen fibers (c), endothelial cells (thick arrows) and smooth muscle cells (s). (Immunofluorescence labeling technique, 400x)


DISCUSSION

Periodontal disease is considered the sixth major complication of diabetes mellitus(33). Despite wide discussion in the literature regarding the biological mechanisms by which diabetes mellitus induce and influence periodontal disease, these mechanisms and their association with the gingival histological alterations needs further clarification.

The metabolic disturbances associated with diabetes can lead to activation of the polyol pathway, high levels of the cytokine TNF-α, the formation of advanced glycation end-products (AGEs), high levels of protein kinase C activation and enhanced oxidative stress (3). The activation of these pathways may be especially important in initiating events linked to inflammation and apoptosis (34, 35).

There are several mechanisms that could be responsible for the higher rate of apoptosis noted in the diabetic patients. One mechanism may be through the cytokine activation of receptors with ‘death domains’, such as TNF receptor-1 (TNF-1) or fas (36). Diabetes is associated with both enhanced TNF and fas/fas-ligand expression (37). Interleukin-1 (IL-1) or interferon-gamma (IFN-γ) may also promote apoptosis, even though their receptors lack death domains, by altering pro-apoptotic gene expression or enhancing production of oxygen radicals (38).

In the present study, strong caspase-3 immuno-reaction has been revealed in the inflammatory cells and in the detected Russell bodies among the en-tire field in the diabetic patients. It has been stated that apoptosis is dependent upon the activation of caspases (39). Caspases are synthesized as catalyti-cally inactive zymogens and must undergo proteo-lytic activation during apoptosis. Initiator caspases, such as caspases 8 and 9, are required to process the executioner caspases, such as caspase- 3, leading to their activation. Active caspase- 3 cleaves over 40 intracellular substrates that cause cell death (40). Alikhani et al. reported that in vivo AGEs induce

fibroblast apoptosis, which is mediated through caspase-3 and signaled through both caspase-8 and caspase-9 activity (41). Interestingly, AGEs stimulate nuclear factor-κβ (NF-κβ) activation, which is anti-apoptotic (42). It should be mentioned that one form of apoptosis in insulin producing cells is indepen-dent of NF-κβ mediated transcription but dependent on caspase-3 activity and poly (ADP-ribose) poly-merase-1 cleavage (43).

Furthermore, Al-Mashat et al. evidenced that the apoptosis enhanced by diabetes plays a functional role in limiting diabetic repair; where the response to the bacterially induced injury is significantly improved through blocking apoptosis with a caspase inhibitor (44). Thus, healing of oral tissues injured by bacterial infection may be impaired, by direct effects on osteoblasts and fibroblasts, or indirectly through promoting inflammation and apoptosis of these matrix-producing cells through diabetes-enhanced caspase-3 activity, leading to higher levels of fibroblast apoptosis and reduced numbers of these cells (45).

Moreover, in the present work we proved the development of Russell bodies in association with Type II diabetes through the use of anti-CD138 antibody (syndecan-1) which was strongly expressed in the diabetic gingiva as compared to the non diabetic group which exhibited negative CD138 immunoreactivity. Syndecan-1 (CD138), a member of the transmembrane heparan sulfate proteoglycan family, acts as an extracellular matrix receptor (46,47) and is involved in many cellular functions, including cell-cell adhesion and cell-matrix adhesion. Expression of syndecan-1 typically is observed on the surface of mature epithelial cells; however, stromal expression also might be seen in developing tissues. Within the hematopoietic system, syndecan-1 expression is thought to be restricted to normal and malignant plasma cells (48,49). It should be mentioned that the hyaline substance detected in the Russell bodies corresponds in particular to the


accumulation of immunoglobulins in the cytoplasm of plasma cells within the endoplasmic reticulum cisternae and they may exist as smaller particles in extracellular locations as well (21-23)

.

Previous studies reported that Mott cells have been localized, although usually in small numbers, in the lymphoid organs of man and laboratory animals, and in increased numbers after hyperimmunization as well as in association with chronic inflammatory lesions, lymphocytic malignancies and acquired immune deficiency syndrome (AIDS). Mott cells appear also in association with genetically determined autoimmune diseases of mice. Thus, Russell bodies, an uncommon finding that may be associated with chronic inflammatory conditions (50).

T-cells might promote the formation of Russell bodies (Mott cells) through the release of soluble factors, such as IL-2 (27). However, the precise mechanism by which T-cells affect Mott cell formation is presently unknown.

In the ongoing study, strong IL-2 immunoreaction has been detected in the inflammatory cells and Russell bodies of the diabetic group. This has been further supported by the histomorphometric analysis data which revealed a high significant increase in the area percentage of IL-2 immunoreaction in the gingiva of diabetic group in comparison to the control group. In accordance, Rabinovitch and Suarez-Pinzon reported that in diabetic patients , there is an immunoregulatory disorder where a T helper 1 (Th1) subset of T cells and their cytokine products; IL-2, IFN-γ and TNF-β, dominate over an immunoregulatory (suppressor) T helper 2 (Th2) subset of T cells and their cytokine products; Interleukin 4 (IL-4) and Interleukin 10 (IL-10). This allows Type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islets (insulitis) and culminating in beta-cell destruction. Type 1 cytokines activate 1) cytotoxic T cells that interact specifically with beta-cells and destroy

them, 2) macrophages to produce proinflammatory cytokines (IL-1 and TNF-α), 3) oxygen and nitrogen free radicals that are highly toxic to islet beta-cells (51).

Furthermore, for conventional T cells, high concentrations of IL-2 can cause activation-induced cell death (AICD), whereas moderate to low concentrations of IL-2 induces effector or memory phenotypes, respectively (52). IL-2 produced by conventional T cells and dendritic cells acts in a paracrine fashion to support the survival and function of Tregs. Type 1 diabetes subjects are reported to exhibit reduced IL-2 production and subsequent Treg dysfunction (53). Phenotypically, these defects include poor maintenance of FoxP3 protein expression, reduced in vitro suppression and increased apoptosis (54).

The fact that diabetics are more susceptible to infections than the non-diabetic (healthy) population suggests that immunologic capability may be deranged in the diabetic population (55,56). In diabetes mellitus, the immunity is deteriorated and the inflammatory process is enhanced in term of increased level of immunoglobulins (57).

In the present work and due to certain limitations, our investigation included the study of IgA and IgM only regardless IgG. However, according to the ongoing results, the levels of these immunoglobulins were elevated in the diabetic group in comparison to the control group.

Previous studies produced conflicting results on immunoglobulins and complement factors in diabetics. Raised levels of IgA, IgG and IgM, reduced level of immunoglobulin classes and complement components were reported in both types of diabetes compared with the controls (58). Type II diabetes is due to complex biochemical disturbances of insulin in action, insulin resistance or both. Some Type II diabetic subjects are hyperinsulinaemic together with high serum glycated proteins/lipoproteins (59). Free insulin and glycated proteins have been found to be immunogenic (60).


Furthermore, compared with controls, serum IgG and IgA were found to be elevated in Type II diabetes (55) whereas IgG and IgM were elevated in Type I diabetics (58). These differences in immunoglobulin classes found in sera of Type I and Type II diabetic subjects might explain differences in the characteristic mouth flora of Type II compared with Type I diabetics with periodontitis(56). This suggests differential immune responses in Type I and Type II diabetes. Recently, Awartani 2010 reported that IgA and IgG levels were found to be significantly higher in poorly controlled Type II diabetes (61).

The high levels of IgM detected in the present study in the diabetic gingiva could be attributed to the fact that serum immunoglobulin levels are dependent on a variety of conditions such as genetic factor, chronic disease and environmental factors. These include ethnic back ground, age, sex, history of allergies or recurrent infections and geographic factors (62).

According to our results, IgA immunoreactivity was detected extracellulary in the gingiva of the diabetic group. Immunoglobulin A is important to control the exposure of the immune system to ingested antigens. The immune system associated with the mucosa produces specific antibodies in order to delay the transport of ingested antigens to circulation. The mechanism is thought to occur by means of IgA antibodies that prevent binding of antigens to epithelial cells. The IgA molecule has the capacity to combine with exogenous antigens, avoid their penetration into circulation and trigger an immunologic response. If antigens enter circulation, they stimulate the development of antibodies and cause a cross reaction with tissue-specific self-antigens, possibly resulting in other immunologic events, such as autoimmune diseases (63). Similarly, according to Mathews Russell bodies of various sizes were present intra and extracellulary at inflammatory sites and were positive for immunoglobulin determinants (64).

In conclusion, it has been proved in this study that raised levels of capase-3 in diabetic gingiva is related to IL-2 production which in turn increases Russell bodies formation that is composed partially of accumulated IgM and IgA as evidenced in this work.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Barbara Safiejko-Mroczka for her support and insightful review during preparation of this manuscript.

Conflict of interest

None declared

Ethical approval

Use of human material in this research was in accordance with the Declaration of Helsinki (The World Medical Association Declaration of Helsiniki1964)

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