“Cellular adenomatoid nodules” of the thyroid: Review of 219 fine-needle aspirates

We reviewed 219 consecutive thyroid aspirates previously diagnosed as “cellular adenomatoid nodule.” applying the criteria presented in this paper and reclassifying them as 146 adenomatoid nodules (AN), 31 cellular adenomatoid nodules (CELL-AN), 29 cellular adenomatoid nodules vs. follicular neoplasms (CELL-AN vs. FN), 5 follicular neoplasms (FN), 2 FN of oxyphilic cell type, 4 papillary carcinomas, and 2 chronic lymphocytic (Hashimoto's) thyroiditis. Of the 146 adenomatoid nodules, 14 occurred in multinodular goiters on histologic examination. Of the 31 CELL-AN, five had surgery: two were follicular adenomas, one was papillary carcinoma, and two were multinodular goiters. Of the 29 CELL-AN vs. FN, 11 had surgery: six were follicular adenomas, two were follicular carcinomas, two were multinodular goiters, and one was Hashimoto's thyroiditis. Surgery on four FN showed follicular adenomas (a fifth patient was lost to follow-up). Of the two FN of oxyphilic cell type, one was a multinodular goiter and the other a follicular adenoma with oxyphilic cells. Three of the four papillary carcinomas were confirmed histologically (one patient was lost to follow-up). Of the two cases showing Hashimoto's thyroiditis, one was diagnosed as FN on repeat aspiration and follicular carcinoma at surgery. After review, we identified 40 patients at higher risk of harboring a neoplasm and 31 with cellular adenomatoid nodules. Twenty-five underwent surgery with the above results. In the classification of follicular lesions of the thyroid by FNA, adherence to strict cytologic criteria helps identify those patients who will benefit most from surgery.     

CA 19-9 expression in subacute (de Quervain's) thyroiditis: an immunohistochemical study.

Carcinoma antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA) expression was immunohistochemically investigated in 48 cases of subacute granulomatous (de Quervain's) thyroiditis, two of focal lymphocytic thyroiditis, three of Hashimoto's thyroiditis, two of Graves' disease, and seven follicular adenomas, 27 follicular carcinomas, and eight papillary carcinomas of the thyroid. CA 19-9 expression was found in all cases of subacute thyroiditis, lymphocytic thyroiditis, and papillary carcinomas examined and in approximately 50% of follicular adenomas and carcinomas. The strongest CA 19-9 staining was demonstrated in late stage subacute thyroiditis and in papillary carcinomas with marked sclerosis. Occasionally CA 19-9 expression was present in seemingly normal thyroid parenchyma adjacent to the thyroid lesions investigated. CEA was found in the center of the granulomatous lesions in acute stage subacute thyroiditis. All neoplasms were CEA negative. CA 19-9 and CEA could be demonstrated occasionally in multinucleated giant cells of subacute thyroiditis, which may suggest that these giant cells are of either histiocytic or follicular cell origin. Immunohistochemical investigation with antibodies against CA 19-9 and CEA may help to histomorphologically define subacute granulomatous thyroiditis.     

Proliferative activity of oxyphilic (Hurthle) cells in reactive and neoplastic thyroid lersions

The proliferative potential of oxyphilic (Hurthle) cells (HCs) present in neoplastic and non-neoplastic thyroid lesions is uncertain. To estimate the HCs ability to proliferate and to determine whether their proliferative activity correlates with the biologic behavior of different thyroid oxyphilic lesions, we selected 31 cases of chronic lymphocytic (Hashimoto’s) thyroiditis and 28 oxyphilic (Hurthle cell) thyroid tumors, including 12 adenomas and 16 carcinomas. Seven histologically normal thyroid specimens from euthyroid patients served as control tissue. The proliferative activity of HCs was evaluated by means of a double immunostaining for Ki67 and a mitochondrial antigen (which specifically recognizes oxyphilic cells). Oxyphilic cells in thyroiditis had a low proliferative activity (PA: 0.55%), although higher than that of normal thyroid parenchyma (PA: 0.06%). Neoplastic HC lesions had a mean proliferative activity of 1.56% and 6.26% in adenomas and carcinomas, respectively. A statistically significant difference was observed between proliferative activity of non-neoplastic and neoplastic lesions (p<0.01), but not within the tumor group, between adenomas and carcinomas. In addition, HC carcinomas had a statistically significant positive correlation between proliferative activity and tumor size (p<0.01) and the presence of necrosis (p<0.001).     

Hürthle-cell lesions of the thyroid: a combined study using transmission electron microscopy, scanning electron microscopy, and immunocytochemistry

Hürthle cell transformation found in 2 nodular goiters, 2 cases of Hashimoto's thyroiditis, 4 follicular adenomas, 3 follicular carcinomas, 2 papillary carcinomas and 1 anaplastic carcinoma were studied by transmission electron microscopy, scanning electron microscopy and immunocytochemistry. Ultrastructural features of Hürthle cells were identical in non-neoplastic and neoplastic lesions. Cells crammed with mitochondria, showing abnormalities in size, shape and content were prominent in most cases. The presence of distinct smooth-surfaced cells interspersed with cells with many microvilli is almost a pathognomonic scanning electron microscopic feature of benign and malignant Hürthle cell lesions. Most Hürthle cells stained positively for thyroglobulin in all cases, but no immunoreactivity for CEA and calcitonin was found.     

Accumulation of p27(kip1) is associated with cyclin D3 overexpression in the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma

BACKGROUND: The down regulation of protein p27(kip1) (p27) in most cases of thyroid cancer has relevant diagnostic and prognostic implications. However, the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma expresses more p27 than benign oxyphilic lesions do. AIM: To evaluate the mechanism underlying this difference in expression of p27. METHODS: Because high levels of cyclin D3 lead to p27 accumulation in cell lines and clinical samples of thyroid cancer, the immunocytochemical pattern of cyclin D3 in oxyphilic (n = 47) and non-oxyphilic (n = 70) thyroid neoplasms was investigated. RESULTS: In the whole study sample, there was a significant correlation between p27 and cyclin D3 expression (Spearman's r: 0.64; p<0.001). The expression of cyclin D3 and p27 was significantly higher in the oxyphilic variant of follicular carcinomas than in non-oxyphilic carcinomas (p<0.001). In the former, cyclin D3 overexpression and p27 accumulation were observed in a median of 75% and 55% of cells, respectively. In co-immunoprecipitation experiments, the level of p27-bound cyclin D3 was much higher in oxyphilic neoplasias than in normal thyroids and other thyroid tumours. CONCLUSION: These results show that increased p27 expression in the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma results from cyclin D3 overexpression.     

Granulomatous and lymphocytic thyroiditis associated with a follicular adenoma

A 44-year-old woman came to medical attention with right thyroid lobe enlargement and weight loss. Thyroid scan and ultrasound demonstrated a nodule in the right thyroid lobe; a right hemithyroidectomy was performed. Histologic examination documented granulomatous and lymphocytic thyroiditis associated with a follicular adenoma. No inflammation was apparent within the adenoma. The parallels between granulomatous thyroiditis and lymphocytic thyroiditis, including presumed viral initiation and deranged immunologic function, suggest that granulomatous thyroiditis may evolve into chronic lymphocytic thyroiditis; this case may be an example of such a transition. It is proposed that thyroid follicular adenomas have different antigenicity, which may account for the conspicuous absence of inflammation within the tumor.     

Hashimoto's thyroiditis: An update on pathogenic mechanisms,diagnostic protocols,therapeutic strategies,and potential malignant transformation

Hashimoto's thyroiditis, characterized by thyroid-specific autoantibodies, is one of the commonest autoimmune disorders. Although the exact etiology has not been fully elucidated, Hashimoto's thyroiditis is related to an interaction among genetic elements, environmental factors and epigenetic influences. Cellular and humoral immunity play a key role in the development of the disease; thus, a T and B cells inflammatory infiltration is frequently found. Histopathologic features of the disease include lymphoplasmacytic infiltration, lymphoid follicle formation with germinal centers, and parenchymal atrophy. Moreover, the occurrence of large follicular cells and oxyphilic or Askanazy cells is frequently associated to Hashimoto's thyroiditis. Clinically, Hashimoto's thyroiditis is characterized mainly by systemic manifestations due to the damage of the thyroid gland, developing a primary hypothyroidism. Diagnosis of Hashimoto's thyroiditis is clinical and based on clinical characteristics, positivity to serum antibodies against thyroid antigens (thyroid peroxidase and thyroglobulin), and lymphocytic infiltration on cytological examination. The mainstream of treatment is based on the management of the hypothyroidism with a substitution therapy. A relationship between Hashimoto's thyroiditis and a possible malignant transformation has been proposed in several studies and involves immunological/hormonal pathogenic links although specific correlation is still debated and needs to be further investigated with prospective studies.     

A thyroid biopsy with histologic features of both Riedel's thyroiditis and the fibrosing variant of Hashimoto's thyroiditis.

We describe a 36-year-old woman with clinical, laboratory, and histologic features of both Riedel's thyroiditis and the fibrosing variant of Hashimoto's thyroiditis. Features of the former included a hard, fixed thyroid mass and extensive involvement of perithyroidal tissues by dense fibrosis with lymphocytes, histiocytes, and plasma cells. Features supporting Hashimoto's thyroiditis included high serum titers of antimicrosomal and antithyroglobulin antibodies and the histologic findings within the thyroid gland itself: dense fibrous bands dividing the thyroid parenchyma into nodules composed of lymphoid follicles with germinal centers, plasma cells, and oxyphilic metaplasia of follicular epithelial cells. Although Riedel's thyroiditis and the fibrosing variant of Hashimoto's thyroiditis were once considered morphologic variants of the same disease, since the 1970s these diseases have been considered as distinct clinicopathologic entities. The coexistence of both diseases in a patient is rare and is probably coincidental in this instance.     

Thyroglobulin immunostaining in follicular thyroid carcinoma: relationship to the degree of differentiation and cell type

A series of 47 primary and seven metastatic thyroid follicular carcinomas, including well, moderately and poorly differentiated, were tested for thyroglobulin (Tg) using immunohistology. In addition, three combined follicular undifferentiated carcinomas, 17 undifferentiated carcinomas and five renal cell carcinomas metastatic to the thyroid were examined. Only two follicular carcinomas did not stain for thyroglobulin. Some inter-tumour differences in Tg staining were found but there was no absolute correlation between this and the degree of tumour differentiation. The two tumours that failed to stain for Tg were poorly differentiated; thyroglobulin positive poorly differentiated tumours demonstrated a clearly weaker staining pattern for Tg. All but one of 15 oxyphilic follicular carcinomas stained positively for Tg but the staining intensity was often weak. Five of six clear cell follicular carcinomas were positive for Tg but the staining reaction was generally faint and there were often large areas devoid of positive cells. Positive staining was demonstrated in the differentiated areas of combined follicular undifferentiated carcinomas. Undifferentiated carcinomas and metastatic renal cell carcinomas gave negative results. Thyroglobulin is a reliable marker for thyroid follicular carcinoma but the patchy staining pattern, particularly in the less well-differentiated tumours may produce less reliable results in small biopsies.     

Thyroid antibodies are produced by thyroid-derived lymphocytes.

The significance of the characteristic lymphocytic infiltrate in the target organ in organ-specific autoimmune disease is unknown. We have demonstrated the production of thyroglobulin antibodies and immunoglobulins (IgG, IgM and IgA) by thyroid-derived lymphocytes in Graves' disease and Hashimoto's thyroiditis two plaque forming cell (PFC) assays. The thyroid appears to be an important site of thyroglobulin antibody production but the thyroid lymphocytes also contain many IgG PFCs of non-thyroglobulin specificity. Short-term culture and direct thyroglobulin antibody assay on micro-ELISA plates confirmed the results of the PFC assay. Therapies such as carbimazole may therefore be acting on a localized source of autoantibody production.     

The production and characterization of thyroid-derived T-cell lines in Graves' disease and Hashimoto's thyroiditis

Although the thyroid gland itself is a major site of the autoimmune response, the study of T-cell function in autoimmune thyroid disease has usually relied on peripheral blood as a source of cells. In this study, we have established thyroid-derived T-cell lines from six patients with Graves' disease and one patient with Hashimoto's thyroiditis by culturing the thyroid lymphocytes on an autologous thyroid follicular cell monolayer in the presence of exogenous interleukin 2 (IL-2). These T-cell lines have allowed in vitro investigation of thyroid-derived T-cell function, an approach which was previously limited by the number of lymphocytes obtained from the gland. The lines were predominantly OKT3, OKT4, and HLA-DR positive but showed heterogeneous proliferative responses. Some lines gave autologous or allogeneic mixed lymphocyte reactions but other did not. Only one of the seven lines responded well to the thyroid antigens thyroglobulin and microsomes presented by autologous monocytes. However, six of the lines proliferated in the presence of live but not dead autologous thyroid follicular cells, particularly when interferon-gamma (IFN-gamma) was added. This treatment has been shown to enhance HLA-DR and -DQ antigen expression by thyroid follicular cells in vitro. Furthermore, the proliferation induced by IFN-gamma-treated thyroid follicular cells was increased when thyroglobulin was also added. Together these results support the hypothesis that the expression of Ia antigens such as HLA-DR by thyroid follicular cells in autoimmune thyroid disease may be important in enhancing the autoimmune response, conferring on these cells the ability to present thyroid autoantigens to T cells. The use of thyroid-derived T-cell lines should permit a more detailed evaluation of the disordered immuno-regulation in Graves' disease and Hashimoto's thyroiditis than has been possible previously.     

Patterns of basement membrane laminin distribution in nonneoplastic and neoplastic thyroid tissue.

Laminin, a major basement membrane component, is typically absent or partially lost around the epithelial elements of most invasive carcinomas. To evaluate the distribution of laminin in both primary and metastatic thyroid tumors, we studied 14 benign thyroid lesions (eight adenomas, two Graves' disease, two Hashimoto's thyroiditis, one adenomatous hyperplasia, one nodular goiter), 20 carcinomas (seven papillary, six tall cell variant, four follicular, three Hürthle), and eight metastases (five tall cell variant, three follicular) utilizing a polyclonal antibody against highly purified, nidogen-free laminin. All benign lesions showed positive, linear immunostaining along basement membranes. Partial loss or absence of laminin was seen in the solid areas of all types of thyroid carcinomas examined; well-differentiated papillary and follicular tumors, as well as papillary and follicular areas of more poorly differentiated neoplasms, maintained linear laminin immunostaining in the papillary cores beneath the epithelial cells and around follicles. A similar correlation between laminin deposition and architectural organization was seen in metastatic lesions. Hürthle cell carcinomas had a unique fragmented, pericellular immunostaining pattern around individual tumor cells, suggesting uncontrolled laminin synthesis. Our findings suggest that preservation of laminin production in thyroid tumors reflects their degree of differentiation and that absence of laminin correlates with lack of structural organization rather than reflecting invasive and metastatic potential.     

Expression of ras oncogene p21 antigen in normal and proliferative thyroid tissues.

The ras oncogene p21 antigen (p21) has been identified in several epithelial malignancies, including breast, colon, bladder, and prostate. The pattern and intensity of immunoreactivity between normal and neoplastic tissues has been distinctly different. The authors examined thyroid lesions from 73 different cases by immunohistochemistry for the expression of p21 with a monoclonal antibody (RAP-5). Normal thyroid tissues (4) showed the least immunoreactivity, while papillary carcinomas (8), Hurthle cell carcinomas (10), and follicular carcinomas as (3) showed slightly more intense staining than Hurthle cell adenomas (12) or follicular adenomas (9). Anaplastic carcinomas (4) showed much less intense staining than most other carcinomas, while medullary thyroid carcinomas (5) showed only slight immunoreactivity. Inflammatory thyroid lesions associated with goiters, including Hashimoto's thyroiditis (6) and Graves' disease (8), showed moderate to intense expression of p21 as did multinodular goiters (4). Semiquantitative analysis of staining intensity by serial dilution of the primary antibody showed significant differences in staining between normal thyroid and some carcinomas (P less than 0.05), but not between carcinomas and adenomas. These results show that while antibody RAP-5 detects an antigen that is only weakly expressed in normal thyroids, this antigen is more strongly expressed in benign and malignant thyroid tumors, as well as in inflammatory and nonneoplastic proliferative thyroid lesions. It is thus not helpful in identifying differences between neoplastic and non-neoplastic thyroid lesions.     

Immunhistochemical and electron microscope analysis of adenomas of the thyroid gland

Summary Histological, immunhistochemical and electronmicroscopic studies of 12 human, scintigraphically cold, thyroid adenomas with specific cytological differentiation identified four different cell types: oxiphil cell, clear cell, ergastoplasm-rich cell and mitochondrion-rich cell.The oxiphil tumor cell can be recognized light-microscopically by its large size and its eosinophilic granular cytoplasm. Most of these cells do not produce thyroglobulin. The ultrastructural characteristics of oxyphil cells are principally mitochondria in great numbers and many large lysosomes. Clear cell adenomas show a trabecular growth pattern. The tumor cells have an abundance of cytoplasm which contains small acidophilic granules. Immunhistochemically we were able to demonstrate thyroglobulin in small amounts within cytoplasmic granules and more extensively within the follicle lumina. Electronmicroscopically we observed a large number of smooth surfaced vacuoles of varying size, extraordinary large lysosomes and occasional cisternae of rough endoplasmic reticulum, the latter probably corresponding to the immune-histochemically identified thyroglobulin granules. The ergastoplasm-rich-cell adenomas, which to the best of our knowledge have not been previously described, show a predominantly micro-to normofollicular architecture histologically without intrafollicular colloid. The cytoplasm of the ergastoplasm-rich cells reveales a strong positive thyroglobulin-staining reaction. The fine structure of these cells is characterized by the abundance of cisternae of the rough endoplasmic reticulum. The mitochondrion-rich-cell adenomas exhibited a microfollicular structure with an intensive acidophilic granular staining at the basal part of the tumor cells. Immunhistochemically and electronmicroscopically we found some morphologic and functional features which differentiate these cells from the oxyphil cell. Thyroglobulin was located predominantly in the apical portion of the cytoplasm in the mitochondrion-rich cells without sharp demarcation from the luminar thyroglobulin. Electron microscopically fewer basal and laterally located mitochondria were seen in mitochondrion-rich cells compared with oxyphil cells. As we could not find any sign of functional activity in the oxyphilic, clear cell and ergastoplasm-rich cell adenomas we analysed those aspects of the lysosomal system not concerned with the enzymatic digestion of thyroglobulin.     

An immunohistochemical study of calcitonin-containing cells in benign and malignant thyroid lesions

Parafollicular cells (C-cells) in benign and malignant thyroid lesions were studied immunohistochemically with a polyclonal anti-calcitonin (CT) antibody. The C-cells were seen most frequently in the middle third of the lateral lobes in the thyroid gland of normal individuals and patients with Graves' disease and chronic thyroiditis, although in the latter the number of such cells was significantly decreased (p less than 0.05). In adenomatous goiter, C-cells were present in nodular lesions from an early stage of nodule development (frequency about 19%), whereas in the later stage these cells were rarely observed inside type 1 nodules, which were generally characterized by an admixture of follicles with considerably different sizes. However, C-cells were not observed inside type 2 nodules, which were composed of similar-sized follicles, or in the parenchyma of 56 cases of benign and malignant thyroid tumors. These findings suggest that since C-cells are present in nodular lesions, the histogenesis of adenomatous goiter is quite different from that of follicular adenoma; thyroid neoplasms generally contain no C-cells in the parenchyma.     

The pathogenetic connection between Graves' disease and chronic lymphocytic thyroiditis. (The role and incidence of thyroid stimulating antibodies)

Antibody-positivity to thyroid specific antigens (Htg, microsomal) and/or lymphocytic infiltration of the gland's parenchyma were observed in 207 (55%) of 377 patients with Graves's disease. Only in 48 (12.7%) of the cases were the findings in agreement with the criteria of chronic lymphocytic thyroiditis. Human thyroid stimulating antibody (HTSab) was detected in 135 (65%) of these 207 patients. In cases of Graves' disease associated with chronic lymphocytic thyroiditis, this proportion was found to be as high as 89.6% and attained even 100% in cases of Hashitoxicosis (39 patients). The presence of HTSab thus seems to form one of the features of patients with Hashitoxicosis. Infiltrative ophthalmopathy also showed a remarkably high incidence (59%) in this porcess. The typical prevalence of Graves' disease in females in the present material attained a 15:1 female-to-male ratio when the disease was associated with chronic lymphocytic thyroiditis. The results of the present study suggest that chronic lymphocytic thyroiditis associated with Graves' disease promotes the formation of thyroid stimulating antibodies.     

Follicular epithelial dysplasia of the thyroid: morphological and immunohistochemical characterization of a putative preneoplastic lesion to papillary thyroid carcinoma in chronic lymphocytic thyroiditis

In chronic lymphocytic thyroiditis (CLT), the follicular epithelial cells display cytological atypia resembling papillary thyroid carcinoma (PTC), and epidemiological studies have suggested an increased risk of PTC in patients with this condition. While reactive atypia is observed diffusely in CLT-affected thyroid parenchyma, it is not unusual to find microscopic foci morphologically distinct from the surrounding parenchyma, exhibiting more pronounced cytological and architectural atypia. These small atypical lesions, which we term “follicular epithelial dysplasia” (FED), are particularly prominent in cases of severe CLT, yet lack invasive growth, papillary architecture, or intranuclear pseudoinclusions. To gain further insight into their biological significance, we constructed a tissue microarray of 70 cases of CLT, comprised of morphologically normal thyroid, thyroid with reactive atypia, FED, follicular nodular disease (nodular hyperplasia or follicular adenoma), and PTC. Immunohistochemical staining was performed for a marker panel including PTC (HBME-1, cytokeratin 19, galectin-3, and cyclin-D1) as well as TTF-1, thyroglobulin, and p63. Slides were digitally scanned and immunohistochemical staining evaluated using automated image analysis software. FED lesions were positive for TTF-1 and thyroglobulin (50/50, 100 %), though some (13/50, 26 %) also expressed p63. Similar to PTC, strong diffuse staining was observed for HBME-1 (43/50, 86 %), cytokeratin 19 (48/50, 96 %), galectin-3 (20/50, 40 %) and cyclin-D1 (38/50, 76 %). In contrast, normal thyroid, reactive atypia, and follicular nodular disease were negative, or at most, exhibited focal weak staining for HBME-1, cytokeratin 19, and galectin-3. The results of this study demonstrate the presence of atypical microscopic lesions in CLT with an immunohistochemical profile similar to PTC, supporting the concept of a premalignant lesion preceding PTC, arising in the context of severe chronic inflammation.     

Expression of intercellular adhesion molecule-1 (ICAM-1) on thyroid epithelial cells in Hashimoto's thyroiditis but not in Graves' disease or papillary thyroid cancer.

In order to study the possible role of antigen-independent adhesion systems in thyroid autoimmunity, we evaluated by indirect immunofluorescence the expression of lymphocyte functional antigen-1 (LFA-1) and its ligand ICAM-1 on mononuclear cell infiltrates (when present) and thyroid follicular cells of four patients with Hashimoto's thyroiditis, 30 with Graves' disease, five with papillary cancer, two with follicular adenoma, and two normal thyroid specimens. The expression of MHC class I and class II antigens was also evaluated. Most mononuclear infiltrates were LFA-1 positive, as expected. A positivity for ICAM-1 on follicular cells was observed in three out of four Hashimoto's thyroiditis specimens; such a phenomenon was totally absent in Graves' disease or any other pathological condition, or in normal tissue. MHC class II expression on thyrocytes was observed in all the patients with Hashimoto's thyroiditis, in 27 out of 30 with Graves' disease and in three out of five papillary cancer specimens.     

The association between chronic lymphocytic thyroiditis and thyroid tumors

An association between lymphocytic thyroiditis and thyroid papillary carcinoma is still controversial. To assess the relationship, a histopathologic analysis of surgically resected thyroid tumors together with the frequency and severity of chronic lymphocytic infiltration of the thyroid among patients with follicular adenoma, follicular carcinoma, and papillary carcinoma was performed. The prevalence of lymphocytic infiltrate, which is indicative of autoimmune thyroiditis, was significantly higher in patients with papillary carcinoma (58%) than in patients with follicular carcinoma (20%) or follicular adenoma (14%). The lymphocytic infiltration within the tumor compared with the severity of thyroiditis in the nontumorous tissue. Therefore, the association between chronic lymphocytic thyroiditis and papillary carcinoma was confirmed. The possibility that an immunologic mechanism involved in the pathogenesis of papillary carcinoma stimulates lymphocytic infiltration in the thyroid tissue through an autoimmune mechanism is suggested.