S期激酶相关蛋白2及细胞周期抑制蛋白p27在老年甲状腺癌患者癌组织中的表达及临床意义

目的研究S期激酶相关蛋白(Skp)2和细胞周期抑制蛋白p27在甲状腺癌癌组织中的表达及临床意义。方法首次就诊的老年甲状腺癌患者85例、甲状腺腺瘤老年患者40例和结节性甲状腺肿老年患者40例,采用免疫组织化学法检测甲状腺癌、甲状腺腺瘤和甲状腺肿组织中Skp2和p27蛋白的表达情况。结果 (1)甲状腺癌组织Skp2蛋白阳性表达率显著高于甲状腺腺瘤组织和甲状腺肿组织(P<0.001),甲状腺腺瘤组织和甲状腺肿组织Skp2蛋白阳性表达率差异无统计学意义(P>0.05);(2)甲状腺癌组织p27蛋白阳性表达率显著低于甲状腺腺瘤组织和甲状腺肿组织(P<0.001),甲状腺腺瘤组织和甲状腺肿组织p27蛋白阳性表达率差异无统计学意义(P>0.05);(3)Skp2蛋白的表达与肿瘤分期、周围组织侵犯情况及淋巴结转移有关(P<0.05或P<0.01),而与性别和肿瘤大小无关(P>0.05);(4)p27蛋白的表达与肿瘤分期、周围组织侵犯情况及淋巴结转移有关(P<0.05或P<0.01),而与性别和肿瘤大小无关(P>0.05);(5)甲状腺癌组织中Skp2与p27蛋白表达评分呈负相关(r=-0.657,P=0.016)。结论 Skp2和p27蛋白分别在甲状腺癌组织的表达呈负相关,两者参与了甲状腺癌的发生和发展。     

Smad4蛋白在良恶性甲状腺病变组织中的表达及意义

目的探讨Smad4蛋白在良恶性甲状腺病变组织中的表达及意义。方法采用免疫组织化学SP法,对33例甲状腺瘤组织,25例相应甲状腺癌周组织及86例甲状腺癌组织中的Smad4蛋白进行检测。结果 Smad4蛋白阳性表达率在甲状腺癌组织(47.7%,41/86)明显低于癌周组织(88.0%,22/25,P<0.01)和甲状腺瘤组织(72.7%,24/33,P<0.05)。Smad4蛋白阳性表达率与甲状腺癌的组织学类型及分化有关。结论Smad4蛋白低表达可能与甲状腺癌的发生、发展和转移密切相关。Smad4蛋白可以作为评估甲状腺癌预后的重要生物学指标。     

甲状腺癌组织中Smad4蛋白表达变化及意义

目的 探讨甲状腺癌组织中Smad4蛋白表达变化及意义.方法 采用免疫组化ABC法检测在甲状腺癌组织、癌旁正常甲状腺组织中的smad4,并选取CD34标记血管内皮细胞以测定微血管密度(MDV).结果 Smad4在甲状腺癌组织、癌旁正常甲状腺组织中的阳性表达率分别为47.1%、87.5%,两者相比,P＜0.05;MDV在甲状腺癌组织、癌旁正常甲状腺组织中分别为(26.85±8.27)、(8.83±4.94)条/cm2,两者相比,P＜0.01.Smad4蛋白阳性表达率与甲状腺癌的组织学类型、淋巴结转移有关(P＜0.05);Smad4蛋白表达与MDV呈负相关(r=-0.496,P＜0.05).结论 Smad4蛋白在甲状腺癌组织中的低表达可能与肿瘤的发生、浸润与转移有关,可作为预测甲状腺癌预后的重要指标.     

p27、nm23蛋白在甲状腺癌中表达及意义

目的探讨甲状腺癌组织中p27、nm23蛋白表达与其临床病理的关系。方法采用免疫组化SP法检测80例甲状腺良性病变（甲状腺腺瘤和结节性甲状腺肿）和甲状腺癌组织中p27、nm23蛋白的表达。结果p27蛋白在甲状腺良性病变组及甲状腺癌组的阳性表达率分别为85.0%、45.0%（P〈0.01）。nm23蛋白在甲状腺癌组阳性表达率（60.0%）虽高于良性病变组（40.0%）,但差异无统计学意义（P〉0.05）。p27蛋白表达与甲状腺癌的分化程度、淋巴结转移和被膜侵犯程度有关。nm23蛋白表达与甲状腺癌分化程度、被膜侵犯程度有关,而与淋巴结转移无关。结论p27、nm23蛋白异常表达可能在甲状腺癌发生或发展中起一定作用。p27、nm23蛋白的联合检测对甲状腺癌的早期诊断、预后的判断及对制定合理治疗方案有重要的临床应用价值。     

甲状腺癌组织中BAG-1、bcl-2蛋白表达及临床意义

目的 探讨BAG-1、bcl-2在甲状腺癌发生、发展中的作用。方法采用免疫组化法检测48例甲状腺癌、11例甲状腺腺瘤组织中BAG-1、bcl-2蛋白的表达情况，并进行相关性分析。结果BAG-1、bcl-2蛋白在甲状腺癌中的表达率（66．7％、75．0％）均高于甲状腺腺瘤（18．2％、27．3％）：甲状腺癌中两基因的表达呈正相关，BAG-1表达水平在甲状腺癌组织学分型、临床分期及淋巴结转移均有相关性。结论BAG-1特异性表达于甲状腺癌组织中，其可能与bcl-2协同参与甲状腺癌的发生、发展；BAG-1表达状态可作为评估甲状腺癌生物学行为和预后的重要参考指标。     

Smad 4和Galectin-3在胃癌中的表达及其临床意义

目的　研究黏附分子Galectin 3和抑癌基因Smad4在正常组织和胃癌中的表达及其临床意义。方法　采用S P免疫组化法检测Galectin 3和Smad4在胃癌及其邻近正常组织中的表达。所有标本都使用武汉同济千屏HPIAS 2000图像分析软件进行定量分析。结果　Galectin 3在胃癌中的阳性表达率为 85 9%,约有 46%表现出核染色。Galectin 3阳性表达以及染色强度与胃癌的分化程度密切相关(P<0 001),分化越低,染色强度越深,在淋巴结转移的癌组织中表达更明显 (P=0 001),与正常组织比较,具有显著的差异性(P<0 001)。Smad4主要在正常组织细胞浆中表达,在胃癌组织中阳性表达率为 59 36%,随着分化程度降低,表达逐渐减少,染色强度也逐渐减弱,尤其在低分化胃癌中更为明显(P<0 001)。与正常组织相比,Smad4减弱具有明显差异性(P<0 01)。结论Galectin 3表达增强与Smad4蛋白表达减弱都与胃癌的分化程度以及淋巴结转移有关,同时检测,能提高预测胃癌进展程度的准确性,是判断胃癌患者淋巴结转移和预后较好的方法。     

Cyclin D1和β-catenin在甲状腺癌组织中的蛋白表达

目的探讨甲状腺癌中细胞周期素D1（Cyclin D1）和β-catenin蛋白表达及其临床意义。方法采用免疫荧光染色法检测43例甲状腺癌、38例癌旁组织和20例正常甲状腺组织中Cyclin D1和β-catenin的蛋白表达。用SPSS13.0统计软件对Cyclin D1和β-catenin蛋白的表达差异及其与患者的临床病理特征进行统计学分析。结果 Cyclin D1和β-catenin蛋白在甲状腺癌中阳性表达率分别为81.4%（35/43）和88.4%（38/43）,与癌旁组织组织42%（16/38）和34%（13/38）和正常甲状腺组织10%（2/20）和15%（3/20）比较有明显统计学差异（P〈0.05）。在甲状腺癌组织中,Cyclin D1和β-catenin蛋白表达与患者的年龄、性别均无明显统计学差异,而与肿瘤的不同病理类型、临床分期和期淋巴结转移有关（P〈0.05）。结论 Cyclin D1和β-catenin蛋白在甲状腺癌组织中表达明显升高,二者呈正相关,可联合作为评估甲状腺癌临床预后指标。     

Galectin-3与甲状腺恶性肿瘤的研究进展

甲状腺恶性肿瘤从病理学上主要分为乳头状甲状腺癌、滤泡状甲状腺癌、未分化甲状腺癌、Hurthle细胞癌和髓样癌等。研究表明Galectin-3的表达与甲状腺恶性肿瘤有关,其免疫检测对于诊断甲状腺乳头状癌是可靠的,但用于鉴别滤泡癌和滤泡腺瘤时不是一个很灵敏的标记;Galectin-3 mRNA不适合作为甲状腺癌的分子标记。在甲状腺乳头状癌,甲状腺组织Galectin-3蛋白的翻译和加工过程中可能发生了改变,有必要进一步研究。     

STAT3与甲状腺肿瘤

信号转导和转录激活因子(STAT)3是一种癌基因调节蛋白,通过细胞因子受体/Janus激酶(JAK)/STAT信号发挥多重生理学效应.STAT3表达于正常甲状腺及甲状腺肿瘤组织中,研究显示,甲状腺癌中STAT3的表达高于良性肿瘤与正常组织,并且与肿瘤侵袭及淋巴结转移密切相关.可见,STAT3在甲状腺肿瘤的发病机制中具有重要作用.另外发现,STAT3蛋白抑制剂能够抑制甲状腺肿瘤细胞的生长,促进细胞凋亡,并且明显抑制肿瘤细胞的侵袭和迁移,提示STAT3可作为甲状腺癌潜在的治疗靶点.     

存活素与血管内皮生长因子在甲状腺癌中的表达及其意义

目的探讨细胞凋亡抑制因子存活素在甲状腺癌中的表达效应及其与血管内皮生长因子(VEGF)表达的关系。方法采用免疫组织化学方法(SABC法)检测68例甲状腺癌、12例甲状腺腺瘤和10例正常甲状腺组织中存活素、caspase-3、VEGF蛋白表达,并结合甲状腺癌主要临床病理参数进行分析。结果存活素在正常甲状腺组织中未见表达,在甲状腺腺瘤中仅现微弱表达(16.7%),而在甲状腺癌中呈显著表达(57.4%);caspase-3在3种组织均有较高表达(70.0%、75.0%、69.1%),而VEGF在甲状腺癌和腺瘤中有不同程度表达(75.0%和41.7%);甲状腺癌中存活素与caspase-3表达无关,与VEGF表达显著正相关(r=0.302,P<0.05),且二者的表达在甲状腺癌组织学分型、临床分期及淋巴转移组间差异均有统计学意义。结论存活素特异性地表达于甲状腺癌组织中,可能通过阻抗细胞凋亡,并与VEGF协同促进血管生成,参与甲状腺癌的发生发展。     

Galectin-3在甲状腺结节中的表达

目的研究galectin-3在甲状腺结节中的表达.方法应用免疫组化及RT-PCR方法检测甲状腺标本中galectin-3表达.结果无论采用免疫组化或RT-PCR方法,甲状腺癌组织的galectin-3表达率均较高（阳性率为89.7%和91.7%）,不同类型的癌组织中有较大差异,其中乳头状癌和滤泡状腺癌具有较高水平的表达（阳性率100%）.有淋巴结转移者galectin-3表达水平高于无淋巴结转移者.在良性甲状腺结节及正常甲状腺组织,采用免疫组化方法检测的25份甲状腺腺瘤标本和13份结节性甲状腺肿标本仅3例呈阳性,余均为阴性,10份正常甲状腺组织及25份癌旁组织均未发现galectin-3表达.结论galectin-3可作为鉴别良、恶甲状腺疾病的一个较有价值的肿瘤指标.     

Galectin-3 messenger ribonucleic acid and protein are expressed in benign thyroid tumors

Galectin-3 is a protein of the lectin family that has been associated with neoplastic processes in various tissues. In the thyroid, expression of this protein has been described in differentiated follicular cancer, suggesting that the immunohistochemical study of galectin-3 may be a potential marker of malignancy in thyroid neoplasms. The confirmation of these results may represent an extremely useful tool for presurgical diagnosis and medical conduct. In this study, galectin-3 protein and mRNA expression were analyzed in the thyroid tissues from 87 patients with histomorphological diagnosis of multinodular goiter (MNG) (n = 24), follicular adenoma (n = 31), follicular carcinoma (n = 20), papillary carcinoma (n = 12), and five normal tissues. Galectin-3 protein expression was detected by immunohistochemical method in light, fluorescence, and confocal microscopy, using monoclonal antibody. Galectin-3 mRNA expression was detected by the RT-PCR method. Our results showed that the majority of carcinomas expressed galectin-3 protein (follicular, 90%; papillary, 100%). However, in contrast to the previously published data, benign lesions also expressed galectin-3 (adenoma, 45%; MNG, 17%). We further demonstrated by RT-PCR that thyroid tissues with diagnosis of adenoma and MNG-expressed galectin-3 mRNA. Although the galectin-3 immunostaining demonstrated a sensitivity of 93.8% in the identification of cancer, the accuracy in the distinction between benign and malignant tissues was 77.0%. This accuracy was even lower (68.6%) when the galectin-3 expression in follicular adenoma was compared with follicular carcinoma. Thus, the use of galectin-3 immunodetection as a molecular marker for thyroid carcinoma must be interpreted with caution, particularly in the differentiation between thyroid follicular carcinoma and follicular adenoma.     

Human galectin-3 immunoexpression in thyroid follicular adenomas with cell atypia

Human galectin-3 (hgal-3) is a beta-galactoside binding protein involved in a number of physiological and pathological processes. Increasing hgal-3 immunoexpression has been reported in several human tumors, including thyroid carcinomas, but not in benign thyroid lesions. We analyzed the immunolocalization of hgal-3 in cell compartments of benign and malignant thyroid lesions. Hgal-3 immunoperoxidase reaction was carried out on 133 thyroid tissue samples obtained from 113 patients; 20 of these were normal (NT), 85 were benign thyroid lesions [20 colloid nodules (CN), 21 nodular hyperplasias (NH), 7 focal lymphocytic thyroiditis (FLT), 15 Hashimoto's thyroiditis (HT), 22 follicular adenomas (FA)], 25 differentiated carcinomas [15 papillary carcinomas (PC), 6 follicular carcinomas (FC) and 4 Hürthle cell carcinomas (HC)] and 3 anaplastic carcinomas (AC). Among the malignant thyroid lesions, hgal-3 was detected in 12/15 (80%) PC, 3/4 (75%) HC and in 4/6 (66.6%) FC, but in none of the 3 AC. Conversely, hgal-3 immunoexpression was absent in NT and in all benign thyroid lesions, but 1/15 HT and 10/22 (45.4%) FA. In the latter, hgal-3 was mostly expressed in microfollicular areas and in five of the six atypical FA. Hgal-3 cytoplasmic-perinuclear immunolocalization was observed in the majority of thyroid carcinomas and in more than half of the FA, theoretically suggesting an involvement of this protein in thyroid tumorigenesis throughout an antiapoptotic activity. Moreover, hgal-3 expression in FA might anticipate the likelihood of evolution of these benign lesions towards malignancy.     

Molecular profiling distinguishes papillary carcinoma from benign thyroid nodules

Recently we identified a molecular basis for differentiating benign and malignant follicular thyroid tumors. The purpose of these studies was to determine whether molecular analysis can be used to differentiate papillary thyroid carcinomas from benign thyroid nodules. Gene expression patterns of 14 papillary thyroid carcinomas and 21 benign tumors were analyzed by oligonucleotide array analysis. The carcinomas included seven classical papillary thyroid carcinomas (PTC) and seven follicular variant of PTC (FVPTC), and the benign tumors included 14 follicular adenomas and seven hyperplastic nodules. A hierarchical clustering analysis was performed to examine the groups for potential differences. The combined PTC and FVPTC groups had a distinct gene expression profile compared with the benign lesions. The sensitivity for a diagnosis of carcinoma was 93%, with a 100% specificity (one FVPTC clustered with the benign nodules). Cancer gene profiles contained both known (Met and galectin-3) and previously unidentified genes. Gene profiling is a reliable means of distinguishing PTC, FVPTC, and benign tumors of the thyroid. These gene profiles may provide insight into the pathogenesis of papillary thyroid carcinoma and may ultimately enhance the preoperative diagnosis of thyroid nodules on a molecular basis.     

Role of galectin-3 immunodetection in the cytological diagnosis of thyroid cystic papillary carcinoma

OBJECTIVE: Cystic thyroid lesions can harbour an occult papillary carcinoma, which fine needle aspiration (FNA) biopsy may fail to detect. Recently, new markers such as galectin-3 lectin have been proposed to distinguish benign from malignant thyroid lesions of follicular origin. The aim of this study was to assess the role of galectin-3 immunodetection in a series of FNA cytological samples of benign and malignant thyroid cystic nodules. METHODS: We retrospectively analysed galectin-3 expression by immunoperoxidase staining on 32 cytological paraffin-embedded samples of cystic papillary carcinoma and on 12 samples of benign cysts, both obtained by FNA biopsy. Specificity, sensitivity, positive/negative predictive values, and accuracy of standard cytological examination and galectin-3 immunodetection were assessed. RESULTS: Among cystic papillary carcinomas, 29 of 32 samples were galectin-3 positive, whereas standard FNA cytology made a correct diagnosis in only 25 of 32 samples. All the benign cysts were negative for galectin-3. In comparing the sensitivity and specificity of the two methods, it appeared that both had a 100% specificity, whereas the sensitivity of cytological examination alone was 75% versus 89.3% obtained by galectin-3 immunohistochemistry. CONCLUSIONS: Galectin-3 immunostaining represents a valid pre-operative adjunct to pick up malignant cells in those cases where a very poor number of epithelial cells may lead to a cytological misdiagnosis. Therefore, we suggest that in poorly cellular FNA biopsies of simple or complex thyroid cysts, galectin-3 expression by epithelial cells is consistent with a cystic carcinoma and supports surgical treatment indication.     

Polymorphism on codon 98 of the galectin-3 gene is not associated to benign and malignant thyroid tumors

Galectin-3 is a multifunctional protein highly expressed in thyroid cancer. The galectin-3 gene (LGALS3) has several annotated candidates SNPs, however the relationship between galectin-3 SNPs and specific phenotypic variations relevant to health has not been evaluated. In this study, we investigated SNPs in the galectin-3 gene and a putative association with thyroid tumorigenesis. The presence of LGALS3 SNPs in thyroid carcinoma cell lines (NPA, TPC-1, WRO, ARO), thyroid tissues of 55 patients with multinodular goiter or papillary carcinoma diagnosis and lymphocytes of peripheral blood of 45 healthy individuals was evaluated by sequencing and SSCP. The analysis of LGALS3 coding sequence showed that the T98P site presents a great genotypic variation, since we observed both homozygous (AA or CC) and heterozygous (AC) patterns. In thyroid carcinoma cell lines, the genotype of NPA in the LGALS3 T98P site is CC, while TPC-1, WRO and ARO are AC. The genotypic frequency of T98P SNP observed in multinodular goiter (AC= 67%; AA= 23%; CC= 10%) and papillary carcinoma (AC= 68%; AA= 20%; CC= 12%) were similar to the frequency observed in the control population (AC= 60%, AA= 24%, CC= 16%). In conclusion, no association between LGALS3 T98P genotype and the phenotype of the benign or malignant thyroid tumor was observed.     

The investigation of galectin-3 in diseases of the thyroid gland

OBJECTIVE: Malignant tumors of the thyroid gland exhibit a variety of histopathologies and clinical behavior. Immune markers are gaining more and more importance in diagnostic pathology, especially in the differential diagnostics and in the grading of thyroid gland tumors. DESIGN: The Authors investigated the immunohistochemical reaction of galectin-3 (gal3) in patients with various thyroid gland diseases. They tested the diagnostic value of gal3 in determining the benign or malignant nature of various lesions, especially in lesions of follicular origin, because previous results have indicated nearly 100% specificity and sensitivity in this regard. METHODS: Gal3 immunoperoxidase reaction was carried out on 91 sections of thyroid gland samples fixed in formalin and embedded in paraffin. RESULTS: While gal3 expressed itself strongly and diffusely in papillary carcinomas (19 of 20 cases), in other malignant lesions it showed weaker, focal or variable positivity. Focal positivity was found in four of 19 follicular adenomas, and negativity was found in three of 10 follicular carcinomas. In all cases of inflammation a focal positivity was observed (eight of eight cases). All nodular goiter and normal thyroid tIssue were negative (25 of 25 cases). CONCLUSIONS: Based on our results, the gal3 immunohistochemical reaction seems to be reliable in the diagnosis of papillary carcinomas. However, in the case of solitary thyroid nodules and follicular lesions, although it is still a useful supplementary marker, it is not of absolute value (as stated in previous studies) in determining whether a tumor is benign or malignant.     

Determination of galectin-3 messenger ribonucleic Acid overexpression in papillary thyroid cancer by quantitative reverse transcription-polymerase chain reaction

Galectin-3, a lectin-family protein that appears to be involved in malignant transformation, has been reported to be an accurate immunohistochemical marker for thyroid cancer. However, immunohistochemistry is a subjective method that can be difficult to apply to cytologic specimens. Therefore, we sought to develop an objective and quantitative assay to measure galectin-3 mRNA in thyroid tissue to enhance potential clinical use of galectin-3 in the molecular analysis of thyroid nodules. In this study, total RNA from 37 snap-frozen thyroid tissue specimens was isolated from eight papillary and nine follicular thyroid cancers, six follicular adenomas, seven adenomatoid nodules, and seven normal thyroid lobes from patients undergoing thyroidectomy. Normalized levels of galectin-3 mRNA, expressed as picograms per nanogram GAPDH mRNA, were higher in papillary carcinomas (3327 pg/ng) and follicular adenomas (1314 pg/ng) than in thyroid normal tissue (426 pg/ng; P = 0.0012 and 0.032, respectively). Galectin-3 mRNA levels were also higher in papillary cancers than in adenomatoid nodules (P = 0.0012). However, galectin-3 mRNA levels were not statistically greater in follicular carcinomas than either normal tissue or follicular adenomas (P = 0.068 and 0.12, respectively). In summary, in comparison to galectin-3 immunohistochemistry, quantitative measurement of galectin-3 mRNA appears useful in the identification of papillary thyroid cancers (PTCs) but does not appear to be useful in distinguishing follicular carcinomas from follicular adenomas.     

Heparanase, galectin-3, and tissue factor mRNA are expressed in benign neoplasms of the thyroid

Objective: Heparanase, galectin-3, and tissue factor (TF) are overexpressed in solid malignant thyroid tumors. We studied their expression in multinodular goiters (MNGs). Design and Methods: Thyroid tissue specimens from 15 MNGs were obtained during surgery. mRNA expression for galectin-3, heparanase, and TF was assessed by RT-PCR. Results: Isolated expressions of heparanase and galectin-3 mRNA were expressed in 2 and 4 of the 15 MNGs, respectively; 8/15 MNGs were positive for both heparanase and galectin-3. TF mRNA was found in all MNG specimens. Conclusion: Galectin-3, heparanase, and TF RNA expression is prevalent in MNGs. Further studies will be needed to determine the prognostic significance of these findings.     

Serum galectin-1 and galectin-3 levels in benign and malignant nodular thyroid disease

BACKGROUND: Since the histological expression of galectins is increased in thyroid carcinoma, determination of their serum levels may provide useful preoperative information. The goal of this study was to determine if a difference in galectin serum levels could be detected between benign and malignant nodular thyroid diseases. DESIGN: Using validated ELISAs, the concentrations of several galectins were prospectively measured in serum samples from 30 healthy individuals and preoperatively in 90 patients with thyroid disease. Seventy-one patients had multiple thyroid nodules (MTN), 13 patients had a single thyroid nodule (STN), and 6 patients had Graves' disease. Nine of 71 patients with MTN had fine-needle aspiration biopsy (FNAB) of their nodules and in 7 patients a "benign" diagnosis was made, in 0 patient a "malignant" diagnosis was made, and in 2 patients a "suspicious" diagnosis was made. Six of 13 patients with STN had FNAB of their nodules and in 2 patients a "benign" diagnosis was made, in 3 patients a "malignant" diagnosis was made, and in 1 patient a "suspicious" diagnosis was made. RESULTS: Thyroid disease was associated with higher levels of galectins-1 and -3 compared to normal subjects. Using a threshold value of 3.2 ng/mL as a cut-off point, the measurement of serum galectin-3 separated micro- and macropapillary thyroid carcinoma (PAP_CA) from patients with nonmalignant thyroid disease with 74% specificity, 73% sensitivity, 57% positive predictive value, and 85% negative predictive value. Elevated serum galectin-3 concentrations (>3.2 ng/mL) detected 87% of macropapillary thyroid carcinomas and 67% of micropapillary thyroid carcinomas. CONCLUSIONS: Serum levels of galectins-1 and -3 are relatively high in patients with thyroid malignancy but there is considerable overlap in serum galectin-3 concentrations between those with benign and malignant nodular thyroid disease and, to a lesser extent, between those with and without nodular thyroid disease.