Effectiveness of sildenafil and U-74389G in a rat model of colitis.

BACKGROUND: Crohn disease is still incurable. Compounds with anti-inflammatory and/or antioxidative effects are tested in various preclinical models of the disease. Our aim was to investigate the effects of sildenafil and lazaroid U-74389G in an experimental rat model of trinitrobenzenesulfonic acid-induced colitis. MATERIALS AND METHODS: Trinitrobenzenesulfonic acid was instilled into the colon of all male Wistar rats except for the rats belonging to the first group. For 6 days, the animals in group 3 were administered daily sildenafil orally, the rats in group 4 were administered daily U-74389G intravenously, and the rats in group 5 were coadministered daily sildenafil orally and intravenous U-74389G. The rats in groups 1 and 2 were not administered any treatment. During the study, the weights were recorded as a marker of clinical condition. The colon damage was evaluated using macroscopic colon mucosal damage index (CMDI), microscopic (Geboes score), and biochemical methods (tissue tumor necrosis factor [TNF]-α and malondialdehyde [MDA]). RESULTS: Sildenafil reduced TNF-α tissue levels and increased body weight. U-74389G reduced TNF-α, the macroscopic index of mucosal damage score (CMDI) and increased body weight. The combined treatment with sildenafil and U-74389G reduced tissue levels of both TNF-α and MDA, lowered CMDI and microscopic Geboes score, and increased body weight. CONCLUSIONS: U-74389G demonstrated a significant anti-inflammatory activity related to its ability to reduce colonic TNF-α, CMDI score, and improve weight change. We confirmed that sildenafil has anti-inflammatory capacity by reducing colonic TNF-α and by improving body weight. Finally, the combined treatment showed superior effects by reducing colonic TNF-α, colonic MDA, CMDI score, Geboes score, and by improving weight.

Lazaroid U-74389G Administration in Pancreatic Ischemia-Reperfusion Injury: A Swine Model Encompassing Ischemic Preconditioning.

CONTEXT: The potential of lazaroid U-74389G in attenuating injury after ischemia and reperfusion has been reported in various organs. OBJECTIVE: The present study focuses specifically on the pancreas and aims to examine any effects of U-74389G in a swine model of pancreatic ischemia and reperfusion, encompassing ischemic preconditioning. METHODS: Twelve pigs, weighing 28-35 kg, were randomized into two experimental groups. Group A (control group, n=6): Two periods of ischemic preconditioning (5 min each) separated by a 5-min rest interval; then ischemia time 30 min and reperfusion for 120 min. Group B (n=6): the same as above, with U-74389G intravenous injection in the inferior vena cava immediately prior to the initiation of reperfusion. Blood sampling and pancreatic biopsies were conducted at 0, 30, 60, 90 and 120 min after reperfusion. RESULTS: Repeated-measures ANOVA was undertaken to evaluate differences between the two study groups. No statistically significant differences were noted concerning the histopathological parameters in the control and therapy groups (P=0.563 for edema, P=0.241 for hemorrhage, P=0.256 for leukocyte infiltration, P=0.231 for acinar necrosis and P=0.438 for vacuolization). In accordance with the above, serum metabolic data (glucose, creatinine, urea, total and direct bilirubin, total calcium, amylase, lipase, SGOT/AST, SGPT/ALT, ALP, GGT, LDH, CRP, insulin) were not significantly different between the two groups; similarly, tumor necrosis factor-α values (P=0.705) and tissue malondialdehyde levels (P=0.628) did not differ between the two groups. CONCLUSION: This swine model of pancreatic ischemia and reperfusion, encompassing preconditioning, indicates that U-74389G lazaroid does not seem to exert protective effects from pancreatic damage.

The effect of U-74389G on pancreas ischemia-reperfusion injury in a swine model.

BACKGROUND: Oxidative stress is a crucial factor in the pathophysiology of acute pancreatitis and its systemic complications. Lazaroids are a novel class of antioxidants that potently protect pancreatic acinar cells against oxidant attack. The aim of our study was to evaluate the therapeutic potential of 21-aminosteroid U-74389G in pancreatic injury after ischemia and reperfusion of the organ in a swine model. MATERIALS AND METHODS: Twelve pigs (weighing 28-35 kg) were randomized into the following two experimental groups: group A (control group, n = 6): ischemia of pancreas (30 min) followed by reperfusion for 120 min; and group B (n = 6): ischemia of pancreas (30 min), U-74389G intravenous injection (10 mg/kg) in the inferior vena cava, and reperfusion for 120 min. Tissue and blood sampling was conducted at 0, 30, 60, 90 and 120 min after reperfusion. Repeated measures analysis of variance was performed for the evaluation of differences between the two groups. RESULTS: Histopathologic evaluation did not reveal a statistically significant difference concerning hemorrhage (P = 0.193), leukocyte infiltration (P = 0.838), acinar necrosis (P = 0.183), and vacuolization (P = 0.185) in the pancreatic tissue between the two groups; nevertheless, edema seemed to be more pronounced in the U-74389G group (P = 0.020). Serum metabolic data in the control and therapy groups were not significantly different; accordingly, tissue malondialdehyde levels (P = 0.705) and tumor necrosis factor α values (P = 0.863) did not differ between the two groups. CONCLUSIONS: On the basis of the histologic data and the absence of reduction in the malondialdehyde and tumor necrosis factor α levels, it is concluded that the administration of U-74389G does not seem to exert a sizable therapeutic effect in attenuating pancreatic damage from ischemia-reperfusion injury.

Proposal for a 10-high-power-fields scoring method for the assessment of tumor budding in colorectal cancer.

Although tumor budding is linked to adverse prognosis in colorectal cancer, it remains largely unreported in daily diagnostic work due to the absence of a standardized scoring method. Our aim was to assess the inter-observer agreement of a novel 10-high-power-fields method for assessment of tumor budding at the invasive front and to confirm the prognostic value of tumor budding in our setting of colorectal cancers. Whole tissue sections of 215 colorectal cancers with full clinico-pathological and follow-up information were stained with cytokeratin AE1/AE3 antibody. Presence of buds was scored across 10-high-power fields at the invasive front by two pathologists and two additional observers were asked to score 50 cases of tumor budding randomly selected from the larger cohort. The measurements were correlated to the patient and tumor characteristics. Inter-observer agreement and correlation between observers' scores were excellent (P<0.0001; intraclass correlation coefficient=0.96). A test subgroup of 65 patients (30%) was used to define a valid cutoff score for high-grade tumor budding and the remaining 70% of the patients were entered into the analysis. High-grade budding was defined as an average of ≥10 buds across 10-high-power fields. High-grade budding was associated with a higher tumor grade (P<0.0001), higher TNM stage (P=0.0003), vascular invasion (P<0.0001), infiltrating tumor border configuration (P<0.0001) and reduced survival (P<0.0001). Multivariate analysis confirmed its independent prognostic effect (P=0.007) when adjusting for TNM stage and adjuvant therapy. Using 10-high-power fields for evaluating tumor budding has independent prognostic value and shows excellent inter-observer agreement. Like the BRE and Gleason scores in breast and prostate cancers, respectively, tumor budding could be a basis for a prognostic score in colorectal cancer.

Correlations between CCN1 immunoexpression and myocardial histologic lesions in sudden cardiac death.

CCN1 (CYR61) is a member of the CCN family of secreted matricellular proteins; it can regulate the expression of genes involved in angiogenesis and matrix remodeling. The latter mechanisms seem to be of vital importance in the pathophysiology of sudden cardiac death. We performed an immunohistochemical analysis on 62 cardiac tissue specimens derived from individuals of young and middle age who had died of sudden cardiac death. CCN1 immunopositivity was detected in 80.6% of all specimens. Semiquantitative statistical analysis of the staining results revealed that CCN1 immunoreactivity was significantly associated with ischemic morphology and hypertrophy of myocardial fibers, interstitial edema, and atheromatosis of coronary arteries in more than 10% of the myocardial fibers. Taking the previously mentioned correlations into account, ischemia seems to induce myocardial expression of CCN1; therefore, CCN1 immunostaining could be evaluated as a complementary tool in the assessment of ischemic areas when no tissue evidence of necrosis is available.

Survival in patients with stage IV noncardia gastric cancer - the influence of DNA ploidy and Helicobacter pylori infection.

BACKGROUND: Palliative surgery followed by postoperative chemotherapy is a challenging approach in the treatment of stage IV gastric cancer yet patients must be carefully selected on the basis of likely clinical benefit. METHODS: The records of 218 patients with histological diagnosis of gastric adenocarcinoma who underwent palliative surgery followed by postoperative chemotherapy were retrospectively reviewed. Twelve potential prognostic variables including tumour DNA index and serum IgG anti- Helicobacter pylori (HP) antibodies were evaluated for their influence on overall survival by multivariate analysis. RESULTS: The median survival was 13.25 months [95% Confidence Interval (CI) 12.00, 14.50]. Three factors were found to have an independent effect on survival: performance status (PS) [PS 60-70 vs. 90-100 Hazard Ratio (HR) 1.676; CI 1.171-2.398, p = 0.005], liver metastases (HR 1.745; CI 1.318-2.310, p < 0.001), and DNA Index as assessed by Image cytometry (2.2-3.6 vs. >3.6 HR 3.059; CI 2.185-4.283, p < 0.001 and <2.2 vs. >3.6 HR; 4.207 CI 2.751-6.433 <0.001). HP infection had no statistically significant effect on survival by either univariate or multivariate analysis. CONCLUSION: Poor pre-treatment PS, the presence of liver metastasis and high DNA Index were identified factors associated with adverse survival outcome in patients with Stage IV gastric cancer treated with palliative gastrectomy and postoperative chemotherapy. HP infection had no influence on survival of these patients.

Tumor infiltration by FcγRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma.

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan-Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearman's rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6-0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7-0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3-0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64- HLA-DR-myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.

Systematic assessment of protein phenotypes characterizing high-grade tumour budding in mismatch repair-proficient colorectal cancer.

AIMS: A tumour bud is defined as a single tumour cell or tumour cell cluster of up to five cells at the invasive tumour front. Significant differences in survival have been detected in colorectal cancer patients with low- compared to high-grade budding. The aim of this study was to identify potential multi-marker phenotypes characterizing low- and high-grade budding in mismatch repair (MMR)-proficient colorectal cancer. METHODS AND RESULTS: Established and promising prognostic proteins such as epidermal growth factor receptor (EGFR), pERK, RHAMM, RKIP, beta-catenin, E-cadherin, pAKT, p16, p21, Ki67, Bcl-2, vascular endothelial growth factor (VEGF), apoptotic protease activating factor-1 (APAF-1), MUC1, EphB2, matrix metalloproteinase 7, pSMAD2, CDX2, laminin5gamma2 and MST1 were analysed on 208 MMR-proficient colorectal cancers with complete clinicopathological data. The most accurate markers for predicting high-grade budding (more than six tumour buds) were EphB2 (P < 0.001), Bcl-2 (P < 0.001), RKIP (P < 0.001), E-cadherin (P = 0.004), laminin5gamma2 (P = 0.004) and APAF-1 (P = 0.005). On multivariable analysis, only loss of Bcl-2 (P < 0.001) and EphB2 (P < 0.001) were independent predictors of high-grade budding. Bcl-2-/EphB2- tumours were more frequently poorly differentiated (P < 0.001), of advanced pT stage (P = 0.002), lymph node positive (P = 0.023), presented vascular (P = 0.053) and lymphatic invasion (P = 0.005) and had a negative impact on patient survival (P = 0.012). CONCLUSIONS: The multi-marker phenotype EphB2-/Bcl-2- is an independent predictor of high-grade budding and implies increased aggressive behaviour in MMR-proficient colorectal cancer.

Role of tumor size in the pre-operative management of rectal cancer patients.

BACKGROUND: Clinical management of rectal cancer patients relies on pre-operative staging. Studies however continue to report moderate degrees of over/understaging as well as inter-observer variability. The aim of this study was to determine the sensitivity, specificity and accuracy of tumor size for predicting T and N stages in pre-operatively untreated rectal cancers. METHODS: We examined a test cohort of 418 well-documented patients with pre-operatively untreated rectal cancer admitted to the University Hospital of Basel between 1987 and 1996. Classification and regression tree (CART) and logistic regression analysis were carried out to determine the ability of tumor size to discriminate between early (pT1-2) and late (pT3-4) T stages and between node-negative (pN0) and node-positive (pN1-2) patients. Results were validated by an external patient cohort (n = 28). RESULTS: A tumor diameter threshold of 34 mm was identified from the test cohort resulting in a sensitivity and specificity for late T stage of 76.3%, and 67.4%, respectively and an odds ratio (OR) of 6.67 (95%CI:3.4-12.9). At a threshold value of 29 mm, sensitivity and specificity for node-positive disease were 94% and 15.5%, respectively with an OR of 3.02 (95%CI:1.5-6.1). Applying these threshold values to the validation cohort, sensitivity and specificity for T stage were 73.7% and 77.8% and for N stage 50% and 75%, respectively. CONCLUSIONS: Tumor size at a threshold value of 34 mm is a reproducible predictive factor for late T stage in rectal cancers. Tumor size may help to complement clinical staging and further optimize the pre-operative management of patients with rectal cancer.

Apoptotic Markers in the Midbrain of the Human Neonate After Perinatal Hypoxic/Ischemic Injury.

Our previous postmortem studies on neonates with neuropathological injury of perinatal hypoxia/ischemia (PHI) showed a dramatic reduction of tyrosine hydroxylase expression (dopamine synthesis enzyme) in substantia nigra (SN) neurons, with reduction of their cellular size. In order to investigate if the above observations represent an early stage of SN degeneration, we immunohistochemically studied the expression of cleaved caspase-3 (CCP3), apoptosis inducing factor (AIF), and DNA fragmentation by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin 3'-end-labeling (TUNEL) technique in the SN of 22 autopsied neonates (corrected age ranging from 34 to 46.5 gestational weeks), in relation to the severity/duration of PHI injury, as estimated by neuropathological criteria. No CCP3-immunoreactive neurons and a limited number of apoptotic TUNEL-positive neurons with pyknotic characteristics were found in the SN. Nuclear AIF staining was revealed only in few SN neurons, indicating the presence of early signs of AIF-mediated degeneration. By contrast, motor neurons of the oculomotor nucleus showed higher cytoplasmic AIF expression and nuclear translocation, possibly attributed to the combined effect of developmental processes and increased oxidative stress induced by antemortem and postmortem factors. Our study indicates the activation of AIF, but not CCP3, in the SN and oculomotor nucleus of the human neonate in the developmentally critical perinatal period.

Somatic mutations of adenomatous polyposis coli gene and nuclear b-catenin accumulation have prognostic significance in invasive urothelial carcinomas: evidence for Wnt pathway implication.

Wnt pathway signaling is crucial in many cancers and data indicate crosstalk with other key cancer pathways, however in urothelial carcinogenesis it has not been extensively studied. We searched for mutations in adenomatous polyposis coli (APC), a key regulator of the pathway, and studied b-catenin expression and interactions with the expression of other markers of apoptosis, angiogenesis, and proliferation in patients with invasive urothelial cancer. The mutation cluster region of APC was directly sequenced in 70 patients with muscle invasive disease who were treated with surgery and adjuvant chemotherapy. COX-2, p53, Ki67, and b-catenin were studied immunohistochemically and micro vessel density was quantified by CD105 expression. Single somatic amino-acid substitutions (missense) were found in 9 (13%) and frameshift deletions in 2 (3%) tumors, all located in regions adjacent to b-catenin binding sites. Patients having either APC missense mutations or b-catenin nuclear accumulation had less frequent COX-2 overexpression (24% vs. 76%, p = 0.043) and more frequent lymph node involvement (75% vs. 38%, p = 0.023). Patients with either APC mutations or b-catenin accumulation had shorter disease-free interval (13.4 vs. 28 months, p = 0.07), whereas in multivariate analysis they had shorter disease-specific survival (60.5 vs. 20.6 months, p = 0.048). Somatic APC missense mutations are not rare in advanced urothelial neoplasms. Either APC mutations and/or aberrant expression of b-catenin are associated with worse outcome. Further study of the role of the Wnt pathway, potential crosstalk with other pathways and potential candidate therapeutic targets in urothelial cancer is needed.

Evaluation of DNA ploidy in relation with established prognostic factors in patients with locally advanced (unresectable) or metastatic pancreatic adenocarcinoma: a retrospective analysis.

BACKGROUND: Most patients with ductal pancreatic adenocarcinoma are diagnosed with locally advanced (unresectable) or metastatic disease. The aim of this study was to evaluate the prognostic significance of DNA ploidy in relation with established clinical and laboratory variables in such patients. METHODS: Two hundred and twenty six patients were studied retrospectively. Twenty two potential prognostic variables (demographics, clinical parameters, biochemical markers, treatment modality) were examined. RESULTS: Mean survival time was 38.41 weeks (95% c.i.: 33.17-43.65), median survival 27.00 weeks (95% c.i.: 23.18-30.82). On multivariate analysis, 10 factors had an independent effect on survival: performance status, local extension of tumor, distant metastases, ploidy score, anemia under epoetin therapy, weight loss, pain, steatorrhoea, CEA, and palliative surgery and chemotherapy. Patients managed with palliative surgery and chemotherapy had 6.7 times lower probability of death in comparison with patients without any treatment. Patients with ploidy score > 3.6 had 5.0 times higher probability of death in comparison with patients with ploidy score < 2.2 and these with ploidy score 2.2-3.6 had 6.3 times higher probability of death in comparison with patients with ploidy score < 2.2. CONCLUSION: According to the significance of the examined factor, survival was improved mainly by the combination of surgery and chemotherapy, and the presence of low DNA ploidy score.

CD56 as a useful marker in the regenerative process of the histological progression of primary biliary cirrhosis.

OBJECTIVE: Owing to recent contradicting results in the study of the regenerative process after hepatic injury in primary biliary cirrhosis, we investigated the use of CD56 in tissue repair during the histological progression of primary biliary cirrhosis. METHODS: Fifty-three specimens were classified into Ludwig's stages (1-4) as follows: 14 specimens as stage 1, 23 as stage 2, 14 as stage 3, and two as stage 4. Immunohistochemical stain was performed for CD56. The cell types expressing the marker were morphologically analyzed to determine their origin. RESULTS: In normal liver biliary epithelial cells (including the epithelium of terminal bile ducts and bile ductules), hepatocytes, and intermediate cells (features between hepatocytes and biliary cells, distributed in interface between hepatic parenchyma and portal tract) were CD56. In primary biliary cirrhosis specimens, biliary epithelial cells, hepatocytes, and intermediate cells were CD56 distributed as 10 out of 14 cases as stage 1 (71.43%), 18 out of 23 as stage 2 (78.26%), nine out of 14 as stage 3 (64.28%), and two out of two as stage 4 (100%). The total positive cases were 39 of 53 (73.58%). CD56 was expressed equally in all three types of cells. CONCLUSION: These findings indicate that the consistent and uniform expression of CD56 in biliary epithelial cells, hepatocytes, and intermediate cells during hepatic injury in primary biliary cirrhosis is probably related to cellular damage and may be important in tissue regeneration. Furthermore, we cannot distinguish a specific cell type from the three above mentioned ones (biliary epithelial cells, hepatocytes, intermediate cells) as a putative stem cell in primary biliary cirrhosis.

Immunohistochemical expression of estrogen receptors alpha and beta in lobular neoplasia.

The designation lobular neoplasia (LN) of the breast includes atypical lobular hyperplasia and lobular carcinoma in situ. Estrogen receptors (ER) play a significant role in breast carcinogenesis. In the present study, ER-alpha and ER-beta status are evaluated in 30 breast tissue specimens from patients whose main lesion was LN. A standard immunohistochemical procedure, using monoclonal antibodies for ER-alpha and ER-beta, was applied to the lesion and the adjacent normal breast tissues, the latter serving as control. In all cases, both receptors were expressed in LN as well as in normal breast ducts and lobules. Concerning ER-alpha, the Allred score and the percentage of ER-alpha-positive cells were significantly higher in LN than in the adjacent normal breast tissue. On the contrary, regarding ER-beta, the Allred score and the percentage of ER-beta-positive cells were significantly lower in LN compared with normal adjacent breast tissue. Greater increase in the percentage of ER-alpha-positive cells was associated with a smaller reduction in the percentage of ER-beta-positive cells and vice versa (Spearman's rho = -0.5044, p = 0.001). In conclusion, upregulation of ER-alpha and downregulation of ER-beta may represent two discrete molecular events in LN pathogenesis. Of notice, a mutually limiting interaction may exist between the two events.

Immunohistochemical investigation of angiogenic factors in parathyroid proliferative lesions.

OBJECTIVE: The pathological distinction between parathyroid neoplasms and hyperplasias remains difficult in several cases. Endoglin (CD105) is a proliferation-associated and hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells. Vascular endothelial growth factor (VEGF) induces angiogenesis and VEGF-R2 is a tyrosine kinase receptor expressed early in development by endothelial cell precursors. We attempted to examine whether immunohistochemical expression of CD105, VEGF and VEGF-R2 may be useful in distinguishing between parathyroid hyperplasia and neoplasia as well as to elucidate, to some extent, the mechanism of neovascularization in proliferative lesions of the parathyroid gland. DESIGN: Tissue specimens were taken from 38 patients with primary hyperparathyroidism (HPT) (17 adenomas and 21 primary hyperplasias) and from 30 patients with secondary HPT. Normal glands served as controls. METHODS: In a standard immunohistochemical procedure, monoclonal antibodies to endoglin, VEGF and VEGF-R2 were applied to detect angiogenic endothelial cells. Immunostaining was estimated by image analysis and statistical analysis was subsequently performed. RESULTS: Positive CD105 immunoreaction was significantly increased in parathyroid adenomas by comparison with primary hyperplasias (P = 0.033) and with secondary hyperplasias (P = 0.033). When parathyroid adenomas, primary hyperplasia and secondary hyperplasia specimens were comparatively evaluated, VEGF immunoreaction was much more common in adenomas (P = 0.018). In addition, in samples with secondary hyperplasia, VEGF-R2 immunoreactivity was positively linked with VEGF expression as well as with the apoptotic index of parathyroid cells (P = 0.038 and 0.010 respectively). In secondary hyperplasia specimens, an inverse correlation between cyclin D1 immunoexpression and angiogenic indexes, such as CD105 and VEGF, was noticed (P = 0.007 and 0.0017 respectively). CONCLUSIONS: This study shows increased angiogenesis in parathyroid adenomas compared with parathyroid proliferative lesions. In secondarily hyperplastic glands increased angiogenesis and increased apoptosis occur simultaneously; in the latter glands, the overexpression of cyclin D1 does not appear to be the genetic abnormality responsible for increased angiogenesis.

Cytokeratin-7, cytokeratin-19, and c-Kit: Immunoreaction during the evolution stages of primary biliary cirrhosis.

AIMS: The quantitative and qualitative expression of CK-7, CK-19, and c-Kit markers in various cell types were evaluated during the four stages of primary biliary cirrhosis. METHODS: A total of 53 specimens were examined. Thirteen specimens were identified as Ludwig's stage 1, 23 as stage 2, 14 as stage 3, and 2 as stage 4. Immunohistochemical stains were performed for CK-7, CK-19, c-Kit and subsequently graded. The cell types expressing the markers were qualitatively analysed. RESULTS: In normal liver, biliary epithelial cells expressed CK-7, CK-19, whereas the Canals of Hering (CoH) were stained with c-Kit and partly CK-19, contrary to hepatocytes. The aforementioned expression patterns were detected in pathologic samples of PBC, with qualitative and quantitative differences though. CK-7 grading was found to correspond with Ludwig's staging, in contrast to CK-19. c-Kit was absent in the early stages and focally present in the advanced stages. All biliary-type, intermediate cells and hepatocytes were CK-7 positive, particularly in samples with cholestasis, whereas CK-19 was only found in biliary-type and intermediate cells. c-Kit was expressed in CoH which appeared as clusters and strings of cuboidal cells in advanced stages. CONCLUSIONS: CK-7 can be regarded as a histological marker of progression in PBC; CK-19 cannot be assessed as a safe marker in the development of the disease. The absence of c-Kit in the early stages of PBC is related to the destruction of the CoH. CK-7 hepatocyte expression in the advanced stages is likely to be related to damaged hepatocytes' metaplastic potential.

Abruptio placentae and idiopathic arterial calcification in a stillborn.

We report a case of idiopathic arterial calcification in a stillborn of 22 weeks gestational age. The pregnancy was complicated by abruptio placentae and the postmortem examination showed widespread arterial calcification in the placenta, as well as in the pulmonary, renal, cerebellar, and brain arteries.

A common 'aggregation-prone' interface possibly participates in the self-assembly of human zona pellucida proteins.

Human zona pellucida (ZP) is composed of four glycoproteins, namely ZP1, ZP2, ZP3 and ZP4. ZP proteins form heterodimers, which are incorporated into filaments through a common bipartite polymerizing component, designated as the ZP domain. The latter is composed of two individually folded subdomains, named ZP-N and ZP-C. Here, we have synthesized six 'aggregation-prone' peptides, corresponding to a common interface of human ZP2, ZP3 and ZP4. Experimental results utilizing electron microscopy, X-ray diffraction, ATR FT-IR spectroscopy and polarizing microscopy indicate that these peptides self-assemble forming fibrils with distinct amyloid-like features. Finally, by performing detailed modeling and docking, we attempt to shed some light in the self-assembly mechanism of human ZP proteins.

Tissue detection of natural killer cells in colorectal adenocarcinoma.

BACKGROUND: Natural killer (NK) cells represent a first line of defence against a developing cancer; however, their exact role in colorectal cancer remains undetermined. The aim of the present study was to evaluate the expression of CD16 and CD57 [immunohistochemical markers of natural NK cells] in colorectal adenocarcinoma. METHODS: Presence of NK cells was investigated in 82 colorectal adenocarcinomas. Immunohistochemical analysis was performed, using 2 monoclonal antibodies (anti-Fc Gamma Receptor II, CD16 and an equivalent to Leu-7, specific for CD-57). The number of immunopositive cells (%) was evaluated by image analysis. The cases were characterized according to: patient gender and age, tumor location, size, grade, bowel wall invasion, lymph node metastases and Dukes' stage. RESULTS: NK cells were detected in 79/82 cases at the primary tumor site, 27/33 metastatic lymph nodes and 3/4 hepatic metastases; they were detected in levels similar to those reported in the literature, but their presence was not correlated to the clinical or pathological characteristics of the series, except for a negative association with the patients' age (p = 0.031). CONCLUSIONS: Our data do not support an association of NK cell tissue presence with clinical or pathological variables of colorectal adenocarcinoma, except for a negative association with the patients' age; this might possibly be attributed to decreased adhesion molecule expression in older ages.

Multidrug resistance proteins and topoisomerase IIalpha expression in colon cancer: association with metastatic potential.

AIMS: To investigate the role of multidrug resistance proteins and topoisomerase IIalpha in colon cancer. METHODS: Tissue sections from 89 Dukes' stage B-D colon cancer patients were selected. The expression of multidrug resistance proteins and topoisomerase IIalpha in primary tumour cells was assessed by standard immunohistochemistry. The extent of their expression was measured by image analysis and was correlated with clinicopathological features of the patients. RESULTS: P-glycoprotein was associated with the presence of lymph node metastasis (P=0.005), vessel invasion (P=0.0001) and perineural invasion (P=0.020). CONCLUSIONS: P-glycoprotein is probably involved in the processes of local invasion and metastatic dissemination.