Loss of angiotensin-converting enzyme 2 promotes growth of gallbladder cancer.

The aim of this study is to investigate the role of angiotensin-converting enzyme 2 (ACE2) in gallbladder cancer (GBC) and the therapeutic potential of angiotensin receptor blocker in GBC. Human gallbladder epithelial cells (HGBEC) together with GBC cells and tissue samples were used. In vitro studies were carried out to investigate the role of ACE2 in GBC cells. ACE2 levels were studied in in vivo GBC mouse models subject to ARB treatment. ACE2 level was decreased in GBC cells compared with that in normal gallbladder cells. Replenishment of angiotensin II (A2) promoted tumour cell growth, which could be mitigated by ACE2 supplement. ARB blocked A2-induced GBC cell growth and activated ERK. Activity of mTOR was not altered with different ACE2 status. ARB inhibited tumour growth in xenograft mouse models. In vivo study also showed that decreased expression of ACE2 was associated with enlarged tumour size. By genetic replenishment of ACE2 and pharmaceutical use of ARB, restored ACE2 level mitigated GBC growth. Our results supported the rationale for the use of ARB in GBC patients for potential therapeutic benefit.

Inhibition of mTOR pathway attenuates migration and invasion of gallbladder cancer via EMT inhibition.

Gallbladder cancer (GBC) is an aggressive disease in which epithelial-mesenchymal transition (EMT) plays a critical role. Whether inhibition of mTOR effects via EMT reversal in GBC remains unclear. Using genetic and pharmacologic inhibitions of mTOR, we investigated the changes of EMT levels in GBC cells. Expressions of EMT related genes were also studied. Migration and invasion assays were carried out and in vivo tumour metastasis mouse models were established. Circulating tumour DNA was quantified. We used EMT index (ratio of Vimentin/Ecadherin expression) to profile EMT levels. We found that inhibition of mTOR using shRNAs and rapamycin inhibited EMT in GBC-SD gallbladder cancer cells. Inhibition of mTOR inhibited EMT in GBC-SD cells in TGF-ß-dependent manner, which was contributed majorly by mTORC2 inhibition. Rapamycin decreased invasiveness and migration of GBC-SD cells in vitro and in vivo. We have in the current study shown that rapamycin diminishes the ability of invasion and migration of GBC via inhibition of TGF-ß-dependent EMT. Our findings contribute to the understanding of the carcinogenesis of GBC.

The association of polymorphisms on TGFBR1 and colorectal cancer risk: a meta-analysis.

Epidemiological studies found inconsistent results on the association of two variants on TGFBR1 (TGFBR1*6A and Int7G24A) with colorectal cancer (CRC) risk. The present study was aimed to evaluate the association of these two variants with CRC susceptibility via the meta-analysis methods. For variant TGFBR1*6A, nine reports including 6,765 CRC patients and 8,496 unrelated controls were identified. The heterozygotes *6A/*9A showed a significant increased risk of CRC with the pooled OR was 1.12 (95% CI = 1.02-1.23), and the pooled OR for the homozygotes *6A/*6A was 1.13 (95% CI = 0.80-1.58) compared to the homozygotes *9A/*9A. However, under the dominant effect model, the TGFBR1*6A carriers showed a significantly increased CRC risk (pooled OR = 1.12, 95% CI = 1.03-1.23, *6A/*6A and *6A/*9A vs. *9A/*9A). For variant Int7G24A, three case-control studies with 1,074 cases and 1,945 controls were found. Although no significant association was found for heterozygosity Int7G24A carriers with CRC risk (pooled OR = 0.97, 95% CI = 0.67-1.42), the homozygosity A/A carriers showed a significant elevated risk of CRC (pooled OR = 1.68, 95% CI = 1.14-2.47) compared to G/G homozygotes. Under the recessive effect model, homozygotes A/A showed a 71% increase of CRC risk compared to the A/G and G/G genotype carriers (pooled OR = 1.71, 95% CI = 1.17-2.51). These data strongly suggested that the two polymorphisms of TGFBR1 may confer low-penetrance susceptibility of CRC risk.

Inhibition of arterial allograft intimal hyperplasia using recipient dendritic cells pretreated with B7 antisense peptide.

BACKGROUND: Low expression or absence of dendritic cell (DC) surface B7 molecules can induce immune tolerance or hyporesponse. Whether DCs could induce indirect allogeneic-specific cross-tolerance or hyporesponse to recipient T cells remains unclear. METHODS: Generated from C3H/He mice bone marrow cells pulsed with donor antigen from C57BL/6 mice, recipient DCs were incubated with B7 antisense peptide (B7AP). Immune regulatory activities were examined in vitro by a series of mixed lymphocyte reactions. Murine allogeneic carotid artery orthotopic transplantation was performed from C57BL/6 to C3H/He. Recipients were given B7AP-treated DCs 7 days before transplantation. Allograft pathological analysis was done 2 months after transplantation. RESULTS: B7AP-pretreated DCs markedly inhibited T-cell proliferation compared with untreated group. Pretreated T cells exhibited markedly reduced response to alloantigen versus third-party antigen. Pathological analysis of arterial allografts demonstrated significant reduction of intimal hyperplasia in B7-AP pretreated group versus control. CONCLUSION: Blockade of B7 molecules by B7AP could induce indirect allogeneic-specific hyporesponse and inhibit arterial allograft intimal hyperplasia, which may be involved in future strategies for human allograft chronic rejection.

Osteopontin and integrin are involved in cholesterol gallstone formation.

BACKGROUND: This study aimed to investigate the role of osteopontin and its receptor, integrin αv, in gallstone formation using human tissue specimens and a guinea pig lithogenic model. MATERIAL/METHODS: The nucleation role of osteopontin was determined in patients' and normal gallbladder bile samples in vitro. Normal gallbladder was the control, and gallstone gallbladders were divided into group I (with normal epithelia) and group II (with degenerated epithelia) based on pathology change. Immunostaining, mRNA and protein expressions of osteopontin and integrin αv were analyzed. The animals were randomly divided into a lithogenic diet group and a normal diet group; the osteopontin mRNA expression in gallbladder and liver and osteopontin concentrations were determined. RESULTS: Osteopontin prolonged nucleation time and inhibited the pro-nucleating role induced by calcium in human bile in vitro. Immunostaining for osteopontin and integrin αv in human gallbladder tissues showed a higher reactivity in Group I than control group and Group II. The immunostaining in Group II was weaker than control group; similar results were observed for mRNA and protein expression of osteopontin and integrin αv. In the animal assay, the mRNA expression and concentration of osteopontin in gallbladder and liver gradually increased at initial stages and decreased in later stages. The concentrations of osteopontin in bile and serum of guinea pig showed similar trends. CONCLUSIONS: Our results suggest that osteopontin is involved in cholesterol gallstone formation, and the role of osteopontin might correlate with integrin αv and calcium.

A randomized trial of somatostatin to regulate the VEGFs/VEGFRs in patients with gastric cancer.

BACKGROUND/AIMS: Angiogenesis and lymphangiogenesis are essential for tumor growth and metastasis. Vascular endothelial growth factors and their receptors (VEGFs/VEGFRs) are important to modulate vasculogenesis. Disregulation of the VEGFs/VEGFRs are closely related to tumor progression and prognosis. METHODOLOGY: We established an open-label, randomized trial to assess the effect of somatostatin on the patients with primary gastric cancer and total gastrectomy. All the 60 patients were randomly divided into somatostatin pretreatment and placebo groups and we used ELISA, real-time PCR and immunohistochemistry analysis to identify the level of VEGFs. RESULTS: The results showed that the patients pretreated with somatostatin had a significant decrease in serum VEGF level, but not in the tissue mRNA level, and the decrease in serum VEGF was partially dependent on the synthesis and degradation of the protein but not the transcription of mRNA. We also found the tissue Flt-4 (VEGFR-3) protein decreased in somatostatin pretreatment group. CONCLUSIONS: We concluded that somatostatin exerts its anti-vasculogenesis effect by downregulating the serum VEGFs and Flt-4 level. Somatostatin can be used as an important adjuvant to improve the survival of gastric cancer patients.

Bibliometric analysis of pressure ulcer research: 1990-2009.

PURPOSE: Bibliometric evaluation provides a method for evaluating trends in publications related to a specific topic. This search focused on pressure ulcer (PU) care; journals and authors with the greatest contribution and impact were emphasized. DESIGN: Bibliometric evaluation. METHODS: Data encompassing the period from 1990 to 2009 were extracted from the Science Citation Index online version. We analyzed selected documents with "pressure ulcer" as a part of the title, abstract, or key words and reported the following parameters: trends of publication output, document types, subject category, journal pattern, authorship, and research in nursing disciplines. RESULTS: The annual number of articles on PUs grew at a rapid rate, from approximately 39 in 1991 to 259 in 2009. The main subject categories in which research on PUs was conducted were surgery and nursing, each of which accounted for more than 10% of total articles. The United States was the dominant country in terms of volume of articles. The relationship between nurse staffing and PU-related outcomes is currently the major focus of PU nursing research, followed by risk assessment scale evaluations. CONCLUSION: We found that the number of PU-related publications has grown at a rapid rate over the past 20 years, reflecting an increasing awareness of the importance of PU prevention and management.

Effects of selenium on the antioxidant enzymes response of Neocaridina heteropoda exposed to ambient nitrite.

The effects of dietary Selenium (Se) supplementation on muscle superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activities and haemolymph superoxide anions (O(2)-) of Neocaridina heteropoda exposed to ambient nitrite were investigated. The results showed supplementation of Se in diet could enhance the resistance of shrimp to low concentration ambient nitrite. The results demonstrated that Se might have a potentially useful role as an effective antioxidant and resistance to aqueous nitrite in shrimp and the effect of the organic Se was better than that of the inorganic Se.

Over-expression of c-FLIP confers the resistance to TRAIL-induced apoptosis on gallbladder carcinoma.

Gallbladder carcinoma (GBC) is an aggressive malignancy with high mortality, mainly due to the reduced chance of curative resection and the resistance to chemotherapeutic drugs. Here, we showed that cellular Fas-associated death domain-like interleukin-1 converting enzyme inhibitory protein (c-FLIP), an anti-apoptotic protein, was over-expressed in the most of gallbladder carcinoma tissues, as judged by immunohistochemistry. Semi-quantitation was performed by determining the percentage of c-FLIP-positive cells: no positive cells (-), approximately 1% positive cells (+), approximately 30% positive cells (++), and >70% positive cells (+++). Out of the 35 tissue specimens of gallbladder carcinoma, positive c-FLIP expression was found in 26 samples (6/positive+++, 13/++, 7/+), whereas negative or weak c-FLIP staining was detected in normal (1/+, 9/-) and adenomatous (2/+, 8/-) gallbladder tissues. Then, we used a small interference RNA (siRNA), which can substantially down-regulate the expression levels of c-FLIP mRNA and protein in GBC-SD and SGC-996 human gallbladder carcinoma cells, as confirmed by real-time PCR and western blot analyses. Furthermore, the combined treatment with the c-FLIP siRNA and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) significantly induced apoptosis in gallbladder carcinoma cells, as judged by the increases in pyknosis, caspase-3/7 activities, and Annexin V-propidium iodide labeling, a marker for chromatin condensation. Thus, the siRNA-mediated down-regulation of c-FLIP profoundly enhances the sensitivity to TRAIL-induced apoptosis. In conclusion, c-FLIP expression is up-regulated in gallbladder carcinoma and the down-regulation of c-FLIP sensitizes TRAIL-induced apoptosis. The present study provides a potent strategy for the treatment of gallbladder carcinoma by targeting the c-FLIP.

Effect of Lidan Granule on bile lithogenesis in patients with choledocholithiasis combined with cholecystolithiasis.

OBJECTIVE: To observe the effect of Lidan Granule (, LDG) on bile lithogenic tendency and biliary 33.5 kd vesicular protein (VP) and to explore its mechanism. METHODS: Sixty patients with choledocholithiasis combined with cholecystolithiasis were randomly assigned to the LDG treated group, the sodium cholate treated group for positive control, and the untreated control group, 20 patients in each group. The 4 bile lithogenic trend indexes, including lithogenic index (LI), unconjugated bilirubin percent (UCB%), unconjugated bilirubin saturation index (BSI) and Z-value, were determined before and after treatment. The content of VP in bile was determined as well. RESULTS: Before treatment, the LI, UCB%, BSI and Z-value in the LDG treated group were 1.298+/- 0.265, 34.72+/-2.96, 0.353+/-0.093 and 0.556+/-0.499, respectively, which was decreased after the 2-week treatment to 0.926+/-0.208, 8.93+/-1.19, 0.154+/-0.056 and 0.257+/-0.211, respectively (all P<0.05). Meantime, the content of VP was also lowered from 0.050+/-0.005 g/L to 0.032+/-0.005 g/L. However, no significant change in any of the above-mentioned indexes was found in the other two groups. CONCLUSION: LDG could effectively suppress bile lithogenic trend and reduce 33.5 kd VP in bile.

[Differential analysis of protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients].

OBJECTIVE: To identify the proteins which play key roles during the formation of cholesterol gallstone, differential analysis was carried out that the proteome of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients. METHODS: Vesicular and micellar phases were isolated by the density gradient ultracentrifugation method. Total proteins from the two phases were extracted, and the protein expressional profiles were established by two-dimensional electrophoresis respectively. The differentially expressed protein spots analyzed by ImageMaster two-dimensional electrophoresis analysis software were identified with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: The concentrations of proteins from vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were (1.5358 +/- 0.0682) mg/ml and (7.1222 +/- 0.2022) mg/ml (P < 0.01) respectively. The average matched protein spots were 120 +/- 24 and 198 +/- 37 in the two groups respectively. There were 72 +/- 16 matched spots in the two representative gels maps and the matched rate was 45.30%. Eight differentially expressed protein spots were identified from the two cholesterol-carrier phases. Among them, 6 were up-regulated with 2 down-regulated in vesicular phase compared with micellar phase. The abundance differentiation of RBP and HSA was confirmed by immunoblotting. CONCLUSIONS: The differential protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were established and 8 differential protein spots were identified successfully. The data may be a basis for further screening the key regulators of formation of cholesterol gallstone.

Bacterial magnetosomes-based nanocarriers for co-delivery of cancer therapeutics in vitro.

In recent times, co-delivery of therapeutics has emerged as a promising strategy for treating dreadful diseases such as cancer. MATERIALS AND METHODS: In this study, we developed a novel nanocarrier based on bacterial magnetosomes (BMs) that co-loaded with siRNA and doxorubicin (DOX) using polyethyleneimine (PEI) as a cross-linker (BMs/DP/siRNA). The delivery efficiency of siRNA as well as the pH-responsive release of DOX, and synergistic efficacy of these therapeutics in vitro were systematically investigated. RESULTS: The structure of DOX-PEI (DP) conjugates that synthesized via hydrazone bond formation was confirmed by 1H nuclear magnetic resonance (NMR). The in vitro release experiments showed that the DP conjugate (DOX-loading efficiency - 5.77%±0.08%) exhibited the long-term release behavior. Furthermore, the optimal BMs/DP/siRNA particle size of 107.2 nm and the zeta potential value of 31.1±1.0 mV facilitated enhanced cellular internalization efficiency. Moreover, the agarose gel electrophoresis showed that the co-delivery system could protect siRNA from degradation in serum and RNase A. In addition, the cytotoxicity assay showed that BMs/DP/siRNA could achieve an excellent synergistic effect compared to that of siRNA delivery alone. The acridine orange (AO)/ethidium bromide (EB) double staining assay also showed that BMs/DP/siRNA complex could induce cells in a stage of late apoptosis and nanocomplex located in the proximity of the nucleus. CONCLUSION: The combination of gene and chemotherapeutic drug using BMs is highly efficient, and the BMs/DP/siRNA would be a promising therapeutic strategy for the future therapeutics.

Osteopontin Deficiency Alters Biliary Homeostasis and Protects against Gallstone Formation.

The precipitation of excess biliary cholesterol as solid crystals is a prerequisite for cholesterol gallstone formation, which occurs due to disturbed biliary homeostasis. Biliary homeostasis is regulated by an elaborate network of genes in hepatocytes. If unmanaged, the cholesterol crystals will aggregate, fuse and form gallstones. We have previously observed that the levels of osteopontin (OPN) in bile and gallbladder were reduced in gallstone patients. However, the role and mechanism for hepatic OPN in cholesterol gallstone formation is undetermined. In this study, we found that the expression of hepatic OPN was increased in gallstone patients compared with gallstone-free counterparts. Then, we observed that OPN-deficient mice were less vulnerable to cholesterol gallstone formation than wild type mice. Further mechanistic studies revealed that this protective effect was associated with alterations of bile composition and was caused by the increased hepatic CYP7A1 expression and the reduced expression of hepatic SHP, ATP8B1, SR-B1 and SREBP-2. Finally, the correlations between the expression of hepatic OPN and the expression of these hepatic genes were validated in gallstone patients. Taken together, our findings reveal that hepatic OPN contributes to cholesterol gallstone formation by regulating biliary metabolism and might be developed as a therapeutic target for gallstone treatments.

Purification and characterization of 33.5 kDa vesicular protein in human bile.

AIM: The present study was undertaken to purify and partially characterize the 33.5-kilodalton (33.5 kDa) vesicular protein in human bile and to explore the possible molecular mechanisms of the initial crystal nucleation process. METHODS: The 33.5 kDa vesicular protein was isolated by ultracentrifugation and further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The purified 33.5 kDa vesicular protein was subjected to N-terminal amino acid sequencing and amino acid analysis. Cholesterol crystallization activity was detected by cholesterol crystal growth assay. The sugar chain of the 33.5 kDa vesicular protein was analyzed by dot-immunobinding assay of lectin coupled to a peroxidase (HRP-DSA, HRP-ConA, HRP-WGA) and was deglycosylated using two different enzymatic approaches (N-deglycosylation and O-deglycosylation) to determine the molecular weight of the protein component, the type of linkage between polypeptide and carbohydrate components. RESULTS: The 33.5 kDa vesicular protein with complicated glycan was an extensively glycosylated (37.3%) monomer and these sugar chains strongly bound to DSA, but did not bind to ConA. Amino acid sequencing indicated that the protein was unique. The 33.5 kDa vesicular protein exhibited potent cholesterol crystallization promoting activity in vitro with derived crystal growth curve indices It, Ig, Ic presented as 0.57, 1.52, and 1.63 respectively. Both enzymatic proteolysis and N-deglycosylation of the protein removed all activity. CONCLUSION: These data suggest the 33.5 kDa vesicular protein may be responsible for the pathogenesis of cholesterol gallstone disease, and the sugar chains play an important role in pro-nucleating process.

Identification of chromosomal imbalances in pancreatic carcinoma using comparative genomic hybridization.

OBJECTIVE: To identify genetic abnormalities in primary pancreatic carcinoma in humans. METHODS: Comparative genomic hybridization (CGH) was used to investigate genomic imbalances in 27 cases of pancreatic carcinomas. Multiple deletions and gains were observed in all tumor specimens. RESULTS: Losses affecting chromosomes 9p, 17p, 4q and 6p and gains involving 8q, 7q, 3q and 1q were commonly observed. CONCLUSIONS: There are multiple regions of chromosomes with changes copy number in pancreatic carcinoma. The altered chromosomal regions may contain several candidate genes which are involved in the development and progress of pancreatic carcinogenesis.

[Inhibition of proliferation of human vascular smooth muscle cells by overexpression of p21 gene].

OBJECTIVE: To observe the apoptosis of human vascular smooth muscle cells (HVSMCs) transfected with p21 gene and investigate its potential mechanism. METHODS: An adenoviral expression vector with full-length cDNA of p21 gene insert (Ad-p21) was constructed and transfected into HVSMC. A mock vector, pAdeno-X-lac Z constructed with the same method, was transfected into HVSMCs as control. Western blotting and DS-PAGE were used to detect the expression of p21 gene. Fluorescence staining was used to observe the changes in nuclei of apoptotic cells and calculate the cell survival rate. Expression of tissue transglutaminase (tTG) in HVSMC was examined by DIG-marked probe. RESULTS: Fluorescence staining showed marked fragmentation of nucleus and pyknosis of chromatin in pAdeno-X-p21 transfected cells. The expression of tTG was at a higher level in pAdeno-X-p21 transfected cells than in normal cells and pAdeno-X-lac transfected HVSMCs. CONCLUSION: Overexpression of p21 gene inhibits the proliferation of HVSMC through apoptosis induced by p21 which may relate with overexpression of tTG.

[The study of pro-nucleating activity on 33.5 x 10(3) vesicular protein in bile].

OBJECTIVE: To evaluate the pro-nucleating activity in 33.5 x 10(3) vesicular protein. METHODS: The model biles were established according Kibe and Zhu. The pro-nucleating activity of 33.5 x 10(3) vesicular protein were examined by polarized light microscopy. The protein and its enzymatic deglycosylation and proteolysis fractions nucleation promoting activity were detected by cholesterol crystal growth assay. RESULTS: 33.5 x 10(3) vesicular protein displayed apparent potency of pro-nucleation with activity of 0.310, and derived crystal growth curve indices It, Ig, Ic were presented as 0.57, 1.52, 1.63 respectively, but after treated by N-glycanase enzyme and pronase, no promoting activity were found. CONCLUSION: The 33.5 x 10(3) vesicular protein may be involved in the nucleation process of gallstone formation, which is regulated by its peptide and sugar chain.

[The study of the pro-nucleating activity of bacteria identified in cholesterol gallstones in model bile systems].

OBJECTIVE: To explore the relationship of bacteria identified in cholesterol gallstones and gallstone formation. METHODS: Observe the bacteria activity in model bile and the influence of bacteria on the cholesterol nucleation time (NT). RESULTS: (1) Model bile were suitable for the growth of E. coli, Pseudomonas aeruginosa, staphylococcus aureus, enterococcus faecalis, clostridium difficile and Clostridium. Propionibacterium acne grew weakly and the growth of Bacteroides fragilis was restrained in model bile. (2) Only pseudomonas aeruginosa and enTerococcus faecalis could ly shorten the cholesterol nucleation time. (3) With pseudomonas aeruginosa or enTerococcus faecalis added in model bile, the formation of cholesterol crystals presented a progressive course of evolution. CONCLUSIONS: Pseudomonas aeruginosa and enterococcus faecalis, not propionibacterium acne, have pro-nucleating ability in model bile.

[ELISA kit for detection of biliary vesicular protein and its preliminary clinical application].

OBJECTIVE: To establish a rapid and precise detective method of 33.5 kd vesicular protein and to screen an effective treatment of cholelithiasis. METHODS: Specific antibody of the biliary vesicular protein was obtained by immunizing rabbits and enzyme-linked immunosorbent assay (ELISA) kit was developed. The concentrations of 33.5 kd vesicular protein in serum and bile of gallstone patients and control were examined respectively. The effects of Cholagogue Dry Syrup and Eulektrol Capsule on decreasing 33.5 kd vesicular protein were also studied by ELISA kit. RESULTS: One-step ELISA equation was Y=0.035 X (r=0.99). The vesicular protein concentrations in serum and bile of cholesterol gallstone group [(179.8+/-97.9) mg/L and (213.4+/-70.1) mg/L respectively] were significantly (P<0.05) higher than in the pigment stone group and control. Data showed that, with 2-week administration, Cholagogue Dry Syrup significantly decreased both biliary and serum 33.5 kd vesicular protein of cholesterol gallstone patients, while Eulekrol Capsule and control groups didn't have the same results. CONCLUSION: The concentrations of 33.5 kd protein are different in cholesterol gallstone patients and healthy groups which might be related to cholesterol nucleation process. Cholagogue Dry Syrup is of cholagogic and litholytic effect by decreasing biliary lithogenesis.

Do hyperechoic thyroid nodules on B-ultrasound represent calcification?

OBJECTIVE: To explore the correlation between hyperechoic thyroid nodules observed on B-ultrasound and histological calcification seen in paraffin-wax sections. METHODS: Records of patients who underwent surgical removal of thyroid nodules diagnosed on preoperative B-ultrasound were analysed retrospectively. Calcification present on B-ultrasound was compared with calcification seen in postoperative pathology specimens. RESULTS: Of the 1,655 patients included in the study, 518 had malignant and 1,137 had benign thyroid nodules. Calcification on B-ultrasound was seen in 366 patients with malignant, and 414 with benign nodules. Calcification was confirmed on histology in 209 and 127 of these patients, respectively, giving a sensitivity and specificity for B-ultrasound in diagnosing calcification (compared with histology) of 95.87% and 47.67%, respectively, in thyroid cancer and 90.71%, and 71.21% respectively in benign thyroid nodules. Microcalcification was seen in 483 patients on B-ultrasound and in 186 on histology, of whom 294 (60.87%) and 152 (81.72%), respectively, had thyroid cancer. CONCLUSIONS: B-ultrasound is a useful and accurate test for detecting calcification in thyroid nodules, with a high sensitivity. There is a close association between calcification (especially microcalcification) and thyroid cancer on both B-ultrasound and pathological examination.