Intensity of Proliferative Processes and Degree of Oxidative Stress in the Mucosa of the Ileum in Crohn’s Disease

The intensity of proliferative processes (estimated from Ki-67 expression) and degree oxidative stress (chemiluminescence assay) in biopsy specimens from the terminal portion of the ileal mucosa were studied in patients with Crohn’s disease. Crohn’s disease is characterized by hyper-regenerative processes in the ileal mucosa. The labeling index (Ki-67 expression) in biopsy specimens from the intact ileal mucosa in patients with the irritable bowel syndrome (reference group) was 10.64 ± 0.62%. The corresponding values in patients receiving monotherapy with mesalazine (group 1) and combination therapy with mesalazine and dalargin (group 2) were 24.05 ± 1.17 and 22.90 ± 0.92%, respectively. Analysis of free radical oxidation showed that this state is accompanied oxidative stress. Spontaneous and H₂O₂-induced luminol-dependent chemiluminescence in biopsy specimens from the ileal mucosa was 1.8-2.3-fold higher compared to the reference group. After therapy, the labeling index in groups 1 and 2 decreased to 18.60 ± 1.18 and 14.38 ± 0.81%, respectively. Histologically, normalization of the disease symptoms was more pronounced after combination therapy. The decrease in free radical oxidation in this group of patients was more pronounced than after mesalazine monotherapy. Our results suggest that oxidative stress plays a role in the hyper-regenerative reaction.     

Correlation of two AgNOR counts with Ki-67 labeling index: A study in fine-needle aspirates of lymphoproliferative disorders and breast carcinoma

The Ki-67 antibody binds to nonresting cells where Ki-67 labeling index (KLI) reflects proliferative activity (PA). The argyrophilic nucleolar organizer regions (AgNOR) counts have been correlated to ploidy and/or PA. Two AgNOR counting methods distinguish ploidy from PA. The first count is the mean AgNOR count (mAgNOR) which correlates with ploidy. The second is the percentage of nuclei with ≥ 5 AgNORs/nucleus (pAgNOR) which reflects PA. To explore this relationship, we separately stained smears of 20 fine-needle aspirates (FNAs) of lymphoproliferative disorders (n = 12) and breast carcinomas (n = 8) for AgNOR and Ki-67. We also double-stained 10 of the smears for both AgNOR and Ki-67. The correlation of pAgNOR counts and KLI was statistically significant (P < 0.0001) whereas it was not between mAgNOR and KLI (P = 0.13). Additionally, using the double stain, the Ki-67 negative cells had an AgNOR granule range of 1-3/ nucleus with a mean of 1.33 (± 1.86 SD). The Ki-67 positive cells showed an AgNOR granule range of 2-12/nucleus with a mean of 4.15 (± 1.02 SD) (P < 0.0002). We thus conclude that pAgNOR is a more reliable indicator of PA than mAgNOR and that four AgNORs/nucleus is an acceptable number differentiating proliferating from resting cells. © 1994 Wiley-Liss, Inc.     

Serum protein alterations in dogs naturally infected with <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>

We conducted this study to describe the serum electrophoretic pattern in dogs associated with the infection of Toxoplasma gondii (T. gondii). The serum protein pattern of 25 dogs with confirmed T. gondii infection and 15 clinically healthy dogs were evaluated using native polyacrylamide gel electrophoresis. Albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin bands were seen from the serum electrophoresis of infected and healthy dogs. Compared to the control group, significant decreases in the mean percentages of albumin (from 46.1 ± 7.2 to 40.8 ± 4.5%, P < 0.05), alpha-1 globulin (from 3.9 ± 0.4 to 0.8 ± 0.2%, P < 0.001), alpha-2 globulin (from 9.0 ± 0.4 to 8.3 ± 0.8%, P < 0.01), and beta globulin (from 18.4 ± 1.2 to 12.1 ± 0.6%, P < 0.001) in the infected group were determined. In contrast, gamma globulin fraction was significantly higher in infected dogs (38.1 ± 4.6%) than in control dogs (22.7 ± 7.2%; P < 0.001). Moreover, significant correlations were determined between the percentages of the albumin and gamma globulin fractions and liver enzyme tests including aspartate aminotransferase and alanine aminotransferase in infected dogs; however, no correlation was observed for the other protein fractions. In conclusion, marked alterations in serum protein pattern associated with strong modifications of serum protein concentrations are in accordance with the hepatic injury as affirmed by liver enzyme tests that were demonstrated in the canine toxoplasmosis. These findings showed that serum protein electrophoresis can be used in the diagnosis and prognosis of canine toxoplasmosis as a supplementary analysis in combination with serological, clinical, and laboratory findings of this disease.     

Cell-cycle analysis detecting endogenous nuclear antigens: Comparison with BrdU-in vivo labeling and an application to lung tumors

The versatility of non-radioactive cell-cycle analysis in detecting endogenous nuclear antigens of the proliferating cells was evaluated. Optimal conditions for immunostaining varied in fixation and pretreatment procedures among antigens, bromodeoxyuridine (BrdU), Ki-67 epitope, DNA polymerase α and PCNA. A significant correlation between BrdU labeling index (LI) was observed in each positive ratio (PR, positive/total neoplastic cells) for nuclear antigens in tumor-sections which had been labeled in vivo with BrdU. The best correlation was observed in Ki-67 PR (y = 1.26x+ 2.5; y= Ki-67 PR; x= BrdU LI; r= 0.97). To determine its prognostic value, Ki-67 analysis was applied to the surgically resected lung tumors. Ki-67 PR were different according to the histologic types of the tumors: 47.8 ± 3.4% in small cell carcinoma; 29.5 ± 3.5% in squamous cell carcinoma; 28.3 ± 4.7% in large cell carcinoma; 15.2 ± 1.8% in adenocarcinoma and 0.1 ± 0.1% in mature carcinoid tumor. When the mean value was used to divide each type to a higher or lower proliferative activity (15% Ki-67 PR for adenocarcinoma and 30% for squamous cell carcinoma), the group with the lower Ki-67 PR showed a significantly more favorable prognosis than that of a higher ratio. Ki-67 PR was not correlated with other pathologic factors such as size, lymph node metastasis or pleural involvement. Non-radioactive cell-cycle analysis was feasible and useful for detecting endogenous nuclear antigens even in the lung tumors, particularly when the analysis was coupled with histologic typing.     

Apoptosis and proliferation in relation to histopathological variables and prognosis in hepatocellular carcinoma

The prognosis of patients with resectable hepatocellular carcinoma depends mainly on the anatomical extent of the tumour and on the general condition of the patient. Given the growing evidence that proliferation indices may be of prognostic significance in hepatocellular carcinomas and that parameters of cell loss (usually, but not exclusively, due to programmed cell death) are biologically relevant, the identification and quantitation of proliferative capacity and apoptosis may be of prognostic importance. In this study four different methods have been used to assess proliferation in a series of 193 curatively (R0) resected hepatocellular carcinomas: mitotic count, immunohistochemical assessment of MIB-1 (Ki-67), proliferating cell nuclear antigen (PCNA), and silver-stained nucleolar organizer regions (AgNORs). Apoptosis was assessed using the in situ-end labelling (ISEL) technique in combination with morphological criteria. Patients who received liver transplantation were excluded. The results obtained were compared with histopathological stage (according to UICC), Edmondson grade, several other histopathological factors, and survival rate. Significant statistical correlations were seen between the mitotic index, the rate of nuclear positivity for MIB-1 and PCNA, and the number of AgNOR dots. In univariate survival analysis, tumour stage and Edmondson grade, mitotic index, MIB-1 and PCNA index, and mean AgNOR number were significant factors influencing patients' survival. On multivariate Cox survival analysis, mitotix index, concomitant cirrhosis, Edmondson grade, and patient age were the only significant independent prognostic factors. Apoptosis was not related to prognosis or to other parameters examined. These results indicated that mitotic index is an additional prognostic parameter which could provide auxiliary information for patients' outcome. MIB-1 and PCNA immunostaining and AgNORs showed a good correlation among themselves. Apoptosis did not predict prognosis in hepatocellular carcinoma. Copyright © 1999 John Wiley & Sons, Ltd.     

Determination of Cell Proliferation Using Mcm2 Antigen and Evaluation of Apoptosis and TGF-β1 Expression in GH-secreting or Clinically Nonfunctioning Pituitary Adenomas

Pituitary adenomas (PA) occasionally show aggressive behavior, with invasion of the surrounding tissues. The identification of markers able to recognize aggressive PA in early stages remains a challenge. We aimed to determine the expression of a new cell proliferation marker, Mcm2, and the presence of apoptosis in PA, and to evaluate the association of clinicopathological features with the apoptotic and proliferative indices. Additionally, the TGF-β1 expression, an inducer of apoptosis, was determined. The proliferative index was determined in GH-secreting or clinically nonfunctioning PA using immunohistochemical (IH) methods for Mcm2 and Ki-67 antigens. The apoptosis was assessed by the TUNEL method and the TGF-β1 expression by IH. A significant positive correlation was found between log Mcm2 index and log Ki-67 index (p < 0.001). Mcm2 and Ki-67 detected a similar number of proliferating cells. Mcm2 index showed a significant association with tumor extension (p = 0.02), but not with tumor invasion. Apoptosis was detected in 17% of the adenomas, with a maximum apoptotic index of 0.77%. Immunoreactivity to TGF-β1 was observed in 77% of the adenomas, showing an association with tumor extension. We concluded that, in this sample, Mcm2 was similar to Ki-67 in the identification of the proliferating cells and that apoptosis was rare.     

Ki-67 expression in primary breast carcinomas and their axillary lymph node metastases: clinical implications

Proliferative activity of tumour cells assessed by immunohistochemical Ki-67 expression is one of several prognostic indicators in breast cancer. The major objective of this study was to investigate the prognostic impact of Ki-67 proliferative activity in the axillary lymph node metastases and in the matched primary breast carcinoma from 194 patients. There was a statistically significant up-regulation of Ki-67 protein in the metastatic deposit compared to where the primary tumour was found (p = 0.001). A low Ki-67 index in both the primary and the metastatic tumours was a favorable prognostic factor. A high index in both primary and metastatic lesion and an up-regulation from a low index in the primary tumour to a high index in the metastatic deposit represented an unfavorable prognostic factor. Multivariate analysis showed that Ki-67 expression in the metastases was a superior independent prognostic factor of clinical outcomes compared to that in the primary tumours. Ki-67 expression in ≥10% of carcinoma cells in the primary tumours and ≥15% in the nodal metastases seems to be optimal cut-off levels. Ki-67 is of value as an independent prognostic factor in breast cancer.     

Expression of proliferating cell nuclear antigen,Ki-67 antigen,estrogen receptor protein,and tumor suppressor p53 gene in cytologic samples of breast cancer: An immunochemical study with clinical,pathobiological, and histologic correlations

Sixty-six unselected breast cancers were analyzed in cytologic smears and histologic sections for the expression of Ki-67, proliferating cell nuclear antigen (PCNA). estrogen receptor protein (ERP), and p53 protein using a standard immunochemical method. The results, expressed as both positive cases and labelling index (LI), were compared with clinical and pathobiological variables. Ki-67 and PCNA immunostaining was seen in all cases, whereas ERP was detectable in 46/63 cases and p53 protein in 20/66 cases. The expression of these markers was generally lower in cytology than in histology, though the differences were not statistically significant. PCNA-LI and Ki-67-LI were closely correlated (P < 0.001), the mean PCNA:Ki-67 ratio being 0.92 ± 0.57. Occasional discrepancies, however, were found. PCNA and Ki-67 expression was associated with an increase in histologic grade and a decrease in ERP content of tumors, whereas p53 was statistically associated with no clinical or pathobiological variables. The data suggest that proliferative activity and oncogene overexpression may be reliably evaluated in breast cancer by FNA cytology, though PCNA is not a suitable indicator for cell proliferation. The results do not resolve the issue as to whether immunostaining for p53 protein constitutes a dedifferentiation product of the tumor, or is a fundamental aspect of the malignant progression. Survival studies in a larger series of tumors are thus needed to elucidate this point. Diagn Cytopathol 1994; 11:131–140. © 1994 Wiley-Liss, Inc.     

In vivo evaluation of the improved MCMS-0102 pacemaker with a rapid pacing mode for induction of experimental heart failure in animals

The MCMS-0102 cardiac pacemaker for rapid ventricular pacing to induce heart failure in animals has been improved in terms of miniaturization and performance. To determine the performance of the new MCMS-0102, six devices were implanted in beagle dogs, and two of these devices were reimplanted for continued pacing in a total of eight beagle dogs. The hearts were paced at 260 beats per minute for 4 weeks (P group: n = 8). The hemodynamic status of the P group was examined and compared with nonpaced dogs (NP group: n = 8). The neurohumoral status of the P group was evaluated before and after rapid pacing. Stable operation of the six devices during rapid pacing was confirmed using the telemetry system. Postmortem examinations revealed features similar to clinical heart failure characterized by massive ascites, pleural effusion, cardiomegaly, and liver congestion in all the paced dogs. Cardiac output was 1.1 ± 0.2 l/min in the NP group and 0.5 ± 0.1 l/min in the P group (P < 0.0001). The left atrial pressure and the central venous pressure of the P group and the NP group were 23 ± 6 versus 6 ± 2 mmHg (P < 0.0001) and 10 ± 3 versus 4 ± 3 mmHg (P < 0.001), respectively. In the paced dogs, plasma renin activity increased from 0.5 ± 0.4 to 8.5 ± 7.4 ng/ml/h (P < 0.05) and atrial natriuretic peptide levels increased from 69 ± 41 to 229 ± 72 pg/ml (P < 0.001). The improved MCMS-0102 was successfully implanted in beagle dogs and it succeeded in inducing the congestive heart failure model.     

Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 in unicystic ameloblastoma

The expression of proliferating cell nuclear antigen (PCNA) and Ki-67 was studied in unicystic and solid ameloblastoma (follicular and plexiform types) using a biotin-streptavidin method on routinely processed paraffin sections. To determine percentage PCNA and Ki-67 labelling indices, positive tumour cells and total tumour cells were counted in areas of each unicystic ameloblastoma corresponding to cystic linings, intraluminal nodules and invading tumour islands, and in solid ameloblastomas. Positive cells in basal and suprabasal layers of cystic tumour lining were also counted with respect to the length of basement membrane determined by image analysis. In unicystic ameloblastoma the invading islands exhibited a significantly higher PCNA labelling index (29.2 ± 16.4%) than intraluminal nodules (13.6 ± 5.4%; P < 0.05). Cystic tumour lining had relatively few PCNA positive cells and a labelling index (5.5 ± 3.3%) significantly lower than invading islands (P < 0.001) or intraluminal nodules (P < 0.003). The labelling indices of solid ameloblastomas of follicular type (48.1 ± 12.9%) were significantly higher than those of cystic tumour lining (P < 0.0001), intraluminal nodules (P < 0.001) and invading islands (P < 0.04) in unicystic ameloblastoma. Similar relationships were found for Ki-67 expression except that comparisons involving invading islands and intraluminal nodules were not significant, a finding probably due to the smaller number of specimens available for quantitative analysis. These results indicate differences in proliferative potential between different areas of unicystic ameloblastoma and between unicystic and solid lesions. The fact that invading tumour islands within the fibrous tissue wall showed high labelling indices is in agreement with the clinical observation that their presence may be related to recurrence after conservative surgery. This provides a biological basis for indicating more radical surgical excision as the treatment of choice for this subgroup of lesions.     

Proliferation not apoptosis as a prognostic indicator in retinoblastoma

 The balance between proliferation and cell death is the major determinant of tumour growth. We analysed the proliferative and apoptotic indices (PI and AI, respectively) of 33 children with retinoblastoma. PI and AI were assessed by immunohistochemistry for Ki-67 antigen and TUNEL staining, respectively. The mean PI was 21.0±21.1%, and higher PI was associated with more advanced tumour stage (P<0.0001) and poor clinical outcome (P<0.05). Patients in whom amplified N-myc oncogene was found (n=6) determined by the multiplex polymerase chain reaction tended to have a higher PI (37.6±27.2%) than those without amplified N-myc (n=27; PI=17.3±18.1). A PI value of over 40% was clearly associated with an unfavourable prognosis. The AI, however, did not correlate with any of the other variables analysed. The findings suggest that proliferation, but not apoptosis, is of critical significance in retinoblastoma biology. PI, as determined by the Ki-67 antigen labelling index, seems to be a relevant histopathological parameter that can predict the clinical outcome of retinoblastoma. Received: 9 November 1998 / Accepted: 24 November 1998     

The Effects of High Dose Pravastatin and Low Dose Pravastatin and Ezetimibe Combination Therapy on Lipid,Glucose Metabolism and Inflammation

Objective Coronary artery disease (CAD) is presently the major cause of mortality and morbidity. Anti-hyperlipidemic treatment is one of the main treatment steps in the management of CAD. Statins are the cornerstones in this treatment. Ezetimibe can be reliably used, when statins prove ineffective in treatment, or to reduce their side effects. In the present study we examined the effects of high-dose pravastatin (40 mg) and low-dose pravastatin (10 mg) + ezetimibe (10 mg) combination therapy on lipid and glucose mechanism, as well as inflammation. Methods This study registered 100 cases. Of the cases, 50 [57.1 ± 11.1 years (24 (48%) females and 26 (52%) males)] were administered 40 mg/day pravastatin (group 1) and 50 [53.2 ± 12.2 years (27 (54%) females and 23 (46%) males)] were administered 10 mg pravastatin + 10 mg ezetimibe (group 2). Results In group 1, total cholesterol fell from 231.1 ± 83.5 mg/dl to 211.3 ± 37.2 mg/dl (p = 0.03), triglyceride from 243.5 ± 96.8 mg/dl to 190.9 ± 55.2 mg/dl (p = 0.003), and LDL cholesterol from 165.7 ± 29.7 mg/dl to 133.4 ± 26.6 mg/dl (p = 0.02). In group 2, total cholesterol dropped from 250.9 ± 51.8 mg/dl to 187.9 ± 34.9 mg/dl (p = 0.001), triglyceride from 270.3 ± 158.9 mg/dl to 154.6 ± 60.7 mg/dl (p = 0.001), and LDL cholesterol from 158.1 ± 47.5 mg/dl to 116.9 ± 26.4 mg/dl (p = 0.001). Insulin resistance decreased from 4.05 ± 2.31 to 3.16 ± 1.90 (p = 0.07) in group 1 and from 2.96 ± 1.50 to 2.05 ± 0.55 (p = 0.009) in group 2. High sensitive C-reactive protein fell from 6.69 ± 6.11 mg/l to 3.02 ± 1.70 mg/l (p = 0.01) in group 1 and from 6.36 ± 2.06 mg/l to 2.68 ± 1.69 mg/l (p = 0.001) in group 2. Conclusion Both therapy regimes are effective. However, we found that low-dose pravastatin and ezetimibe combination therapy is more effective than high-dose pravastatin therapy on lipid metabolism, glucose metabolism and inflammation.     

The Evaluation of Argyrophilic Nucleolar Organizing Region Proteins in Fine-Needle Aspiration Samples of Thyroid

Argyrophilic nucleolar organizing region associated proteins (AgNORs) have been shown to be of interest in a variety of different diseases including thyroid disorders. Our aim was to distinguish benign thyroid lesions from papillary thyroid carcinoma (PTC) via AgNOR count and with a new approach, via AgNOR surface area/total nuclear surface area (NORa/TNa) proportions in the nuclei on fine-needle aspiration (FNA) materials. Thirty patients (eight men and 22 women) whose FNA was compatible with benign lesion and 26 patients (eight men and 18 women) whose FNA was compatible with PTC were included in the study. Fine-needle aspiration materials were stained for AgNOR detection according to a specific protocol. One hundred nuclei per individual have been evaluated, and AgNOR number and NORa/TNa proportions of individual cells were measured and calculated by using a computer program. Patients with PTC had significantly (p < 0.001) higher AgNOR count (4.6 ± 1.2%) than in the patients with benign lesions (2.0 ± 0.5%). Additionally, patients with PTC had significantly (p < 0.001) higher NORa/TNa (13.4 ± 2.4) than in the patients with benign lesion (5.7 ± 1.0). Modified method of AgNOR staining is an easy and reliable method for evaluating proliferation activity of cells in malignant and benign thyroid lesions and it may contribute to routine cytopathology in inconclusive situations.     

Validation of the use of proliferation markers in canine neoplastic and non-neoplastic tissues: comparison of KI-67 and proliferating cell nuclear antigen (PCNA) expression versus in vivo bromodeoxyuridine labelling by immunohistochemistry

In order to evaluate the suitability of Ki-67 and proliferating cell nuclear antigen (PCNA) for determination of proliferative activity, the immunohistochemically determined nuclear expression of these antigens in canine non-neoplastic and neoplastic tissues was compared with the results of in vivo bromodeoxyuridine (BrdU) labelling, which - by measurement of the fraction of S-phase cells - is considered as the standard in the analysis of proliferative activity. The samples investigated consisted of non-neoplastic mammary and lymphoid tissues, and of benign and malignant (primary/metastatic) mammary tumours, and malignant lymphomas. Great regional heterogeneity prevented determination of an overall labelling index (LI) in normal lymphoid tissues. In the remaining combined group of samples, LI values were significantly ranked in the order PCNA>Ki-67>BrdU. However, the correlation of Ki-67 or PCNA as compared to BrdU LI values was only moderate in the combined group [approximately 0.5, Spearman rank test] as well as in most subgroups, whilst it was very poor in the group of primary mammary cancers. These observations indicate that Ki-67 or PCNA LIs as markers of proliferation do not evenly match in vivo BrdU labelling.     

Proliferating activity in columnar cell lesions of the breast

With the introduction of mammographic screening, columnar cell lesions (CCLs) are observed more and more frequently because they are often associated with microcalcifications. Until now, the proliferative activity of these lesions has not been previously evaluated. Ki67 index was performed by immunohistochemistry in CCLs without atypia [columnar cell change (CCC) n = 20 and columnar cell hyperplasia without atypia (CCH without atypia) n = 20], flat epithelial atypia (FEA DIN1A n = 20), low-grade intraductal carcinoma (DIN1C n = 20), high-grade intraductal carcinoma (DIN 2–3 n = 20). Adjacent terminal duct-lobular unit (TDLU) of normal breast tissue served as control. Ki-67 index is extremely low and close in CCLs without atypia (CCC mean 0.1% and CCH mean 0.76%) and paradoxically is lower than in normal TDLU (mean 2.4%) (p < 0.001). In the FEA, in comparison with normal TDLU and CCLs without atypia, the Ki67 is higher (mean 8.2%) (p < 0.001) but extremely close to those of DIN1C (mean 8.9%) (p = 0.6 NS). Lastly, the Ki67 index is higher in DIN 2–3 (mean 25.4%) than in CCLs without atypia and FEA (p < 0.001). CCLs are disparate lesions having in common cells with columnar configuration but different proliferative characteristics. These data represent findings of biological interest which could help us to better understand these controversial lesions.     

A critical analysis of three quantitative methods of assessment of hepatic steatosis in liver biopsies

The issue of adequately quantitatively evaluating hepatic steatosis is still unresolved. Therefore, we compared three methods of quantitative assessment. Two groups of mice (n = 10 each) were fed standard chow (10% fat, SC group) or a high-fat diet (60% fat, HF group) for 16 weeks, and hepatic triglyceride (HT) and liver tissue were then studied. Paraplast-embedded tissues stained by hematoxylin and eosin (H-E) were compared to frozen sections stained by Oil Red-O (ORO). In addition, the volume density of steatosis (Vv[steatosis, liver]) was measured by point counting (P-C, sections H-E or ORO) or by image analysis (I-A, sections ORO). HT was significantly higher in the HF group (104% greater, P = 0.0004) than in the SC group. With P-C and H-E, Vv[steatosis, liver] was 4.80 ± 0.90% in the SC group and 33.50 ± 3.17% in the HF group (600% greater, P < 0.0001). With P-C and ORO, Vv[steatosis, liver] was 4.86 ± 0.89% in the SC group and 25.21 ± 1.27% in the HF group (420% greater, P < 0.0001). With I-A and ORO, Vv[steatosis, liver] was 4.17 ± 0.85% in the SC group and 23.35 ± 1.58% in the HF group (460% greater, P < 0.0001). Correlations between Vv[steatosis, liver] and HT were strong and significant in all methods. In conclusion, all methods were appropriate and reproducible. In P-C and H-E, there is a slight overestimation of steatosis in the HF animals in comparison to frozen sections and ORO; in frozen sections, differences between P-C and I-A are insignificant.     

Expression of cyclin D1, Ki-67 and PCNA in non-small cell lung cancer: prognostic significance and comparison with p53 and bcl-2

Uncontrolled cell proliferation is the hallmark of malignant tumours. Thus, the proliferative potential of tumour cells is an important prognostic factor. However, evaluation of the prognostic significance of the expression of proteins involved in regulation of cell proliferation remains controversial. In the present study, expression of Ki-67, PCNA and cyclin D1 was estimated in a group of 89 surgically resected non-small cell lung carcinomas using immunohistochemistry. The results were compared with expression of bcl-2 and p53 and with clinicopathological parameters including patients' survival. Ki-67 and PCNA were found to be moderately and highly expressed in 39% and 44% of the tumours, respectively. There was a strong correlation between Ki67 and PCNA expression. Forty five of 88 tumours (51%) showed overexpression of cyclin D1. Surprisingly, cyclin D1 was mainly localized in the cytoplasm and only a small group of tumours (9/88, 10%) showed nuclear staining as well. Bcl-2 and p53 expression was observed in 69% and 30% of the tumours, respectively. All these markers were found to be independent of clinicopathological parameters, except for Ki-67 and bcl-2 expression, which was associated with squamous cell carcinomas. It is concluded that none of the markers that were studied can be used as an independent prognostic factor, whereas the following combinations of markers may have favourable prognostic value: p53 positivity and low Ki-67 expression, p53 positivity and lack of cyclin D1 expression, bcl-2 positivity and low Ki-67 expression, and lack of cyclin D1 expression and low Ki-67 expression.     

Proliferative Potential of Multipotent Mesenchymal Stromal Cells from Human Bone Marrow

We studied the capacity of multipotent mesenchymal stromal cells isolated from human bone marrow (BM) to long-term passaging, cloning, and re-cloning. Initial multipotent mesenchymal stromal cells and cells after gene labeling were studied. Multipotent mesenchymal stromal cells were obtained from donors (13–59 years) and cultured for 7 passages. Third generation lentivector was used for delivery of green fluorescent protein marker gene. The procedure of infection revealed reduced proliferative potential of multipotent mesenchymal stromal cells from elder donors. Hierarchy of precursor cells differing by their proliferative potential was demonstrated in the culture of multipotent mesenchymal stromal cells. Three categories of multipotent mesenchymal stromal cells were identified: mature cells incapable of proliferation (75.7 ± 2.4% population) and cells with low and high proliferative potential (17.6 ± 2.1 and 6.7 ± 0.3%, respectively). The relative content of these cells insignificantly differed from passage to passage. The efficiency of cloning also remains stable, but re-cloning capacity sharply decreased after passage 3 and completely disappeared in multipotent mesenchymal stromal cells after cryopreservation. Thus, cultured multipotent mesenchymal stromal cells represent a heterogeneous and hierarchically organized population and the characteristics of this population depend of the duration of culturing and age of BM donor. This should be taken into account when using multipotent mesenchymal stromal cells in clinical practice.     

Effect of praziquantel prolonged administration on granuloma formation around Schistosoma japonicum eggs in lung of sensitized mice

Schistosomiasis remains a major public health problem and it is an immune disease. The schistosome egg is the primary parasite factor responsible for the overt disease. The eggs release the soluble antigen, which induces intensive tissue reaction, a granulomatous reaction to the eggs. If granuloma formation could be suppressed, overt disease might not develop. Praziquantel is an effective antischistosomal drug especially for adult worms. However, whether praziquantel has a suppressing effect on granuloma formation around schistosome eggs directly remains unclear. The purpose of the present study was to investigate the effect of praziquantel, especially administered persistently, on granuloma formation around Schistosoma japonicum eggs in the lung of sensitized mice. Thirty-six mice were divided into three groups averagely. Group A was a control group. First, the mice were injected with schistosomal eggs hypodermically in abdomen, and 10 days later injected with schistosomal eggs intravenously via a tail vein. Group B was a praziquantel short administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 300 mg/kg for 3 days, from 1 day before the intravenous injection of the eggs. Group C was a praziquantel prolonged administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 150 mg/kg for 5 days weekly until the mice were sacrificed. Three mice of each group were sacrificed on days 7, 14, 28, and 56, respectively after the intravenous injection of the eggs, and the lung tissues were fixed with formalin and the slices were HE stained. The granulomas containing eggs in their centers were selected, and 25–30 granulomas from the animals of each group were measured at each time period. The mean areas of egg granulomas of each group were calculated, and the neutrophilic granulocytes, eosinocytes, lymphocytes, fibroblasts, and macrophages within the egg granulomas were counted. The mean numbers of them of each group were calculated. All the data of each group were analyzed and compared statistically. On day 56 after the intravenous injection of the eggs, the mean area of schistosomal egg granulomas in group B was (227.4 ± 728.0) × 10³ μm², less than that of [(297.9 ± 153.3) × 10³ μm²] in group A, and the suppression rate was 23.7% (P < 0.05). On days 7, 14, 28, and 56, the mean areas of schistosomal egg granulomas in group C were (575.8 ± 155.6) × 10³ μm², (310.5 ± 854.0) × 10³ μm², (267.7 ± 513.3) × 10³ μm², and (214.9 ± 446.4) × 10³ μm², respectively, significantly less than those of [(692.7 ± 232.6) × 10³ μm², (439.4 ± 165.0) × 10³ μm², (385.7 ± 129.3) × 10³ μm², and (297.9 ± 153.3) × 10³ μm²] in group A. The suppression rates were 16.9%, 29.3%, 30.6%, and 27.9%, respectively (P values <0.05). On day 56, the mean numbers of neutrophilic granulocytes were 11.4 ± 5.0 in group A and 5.2 ± 3.1 in group C, respectively, with the suppression rate of 54.4% in group C (P < 0.05). On day 56, the mean numbers of eosinocytes within the egg granulomas were 2.3 ± 2.0, 0.1 ± 0.3, and 0.3 ± 0.6 in groups A, B, and C, respectively, with the suppression rate of 95.7% in group B and 87.0% in group C (P values <0.05). On day 56, the mean numbers of macrophages within egg granulomas were 14.3 ± 6.9 in group C, compared with 18.6 ± 8.2 in group A, the suppression rate was 23.1% (P < 0.05). On day 56, the mean numbers of fibroblasts within the egg granulomas were 6.6 ± 4.4 and 5.8 ± 2.6 in groups B and C, respectively, and compared with 14.3 ± 7.8 in group A, the increasing extents decreased by 53.8% and 59.4%, respectively (P values <0.05). Therefore, the administration of praziquantel, especially the prolonged administration, can suppress the formation of schistosomal egg granulomas, including reduction in the areas of granulomas and suppression of the inflammatory cells and the hyperplasia of fibroblasts within granulomas.