The proliferative activity in oral epithelial dysplasia analyzed by proliferating cell nuclear antigen immunostaining and argyrophilic nucleolar organizer region staining

The proliferative activity of leukoplakia without dysplastic change (LP), epithelial dysplasia (ED), and squamous cell carcinoma (SCC) in the oral mucosa was examined by means of proliferating cell nuclear antigen (PCNA) immunostaining, silver-binding argyrophilic nucleolar organizer region (AgNOR) staining, and the frequency of mitotic figures. Significant differences in the labeling index of PCNA immunostaining (PI) and mitotic index (MI) were noted between LP and ED and between ED and SCC. The mean numbers of AgNORs (AI) significantly differed between ED and SCC. There was a significant positive correlation between PI and MI in samples of ED. However, there was no significant correlation between AI and other indexes. The number and the distribution of PCNA-positive cells in ED varied among samples. Five samples with higher PI and MI indexes than the mean values were selected from those of dysplasia based on the correlation between PI and MI. Their histological features symptomatic of oral ED as defined by the World Health Organization (WHO) Collaborating Centre in 1978, were investigated and compared with 10 samples with lower indexes. Histological findings, such as "loss of polarity of the basal cells," "an increased nuclear-cytoplasmic ratio," "cellular pleomorphism," and "enlarged nucleoli," were significant histological features of these five samples. This study showed that the four histological components described previously and the increased number of mitotic figures used as the index of proliferating activity were the main histological components related to severe ED of oral mucosa. They will provide a useful means of deciding the histopathological grade of oral ED.     

A comparative study of nucleolar organizer region, proliferating cell nuclear antigen and epidermal growth factor receptor staining in colon tumours

The prevalence of hepatitis A virus (HAV) antibody in people has decreased from year to year in Japan. A sequential outbreak occurred in an institution for the mentally handicapped people in Chiba City in the summer of 1995. Eight people were infected including 7 residents and one staff member. We tested to detect antigen in fecal samples by ELISA and PCR for early diagnosis for hepatitis A infection. Four sera and 5 feces were obtained from 5 patients between 2 and 8 days after the onset of symptoms. The anti-HAV IgM was found to be positive in 4 sera examined. The HAV antigen was detected in 3 out of 5 feces using ELISA. An existence of inhibitor in 2 negative specimens against the ELISA was suggested by the recovery test of added antigen. HAV RNA was extracted by CTAB method from feces and detected in 4 our of 5 specimens in PCR amplification and in all of 5 specimens in nested PCR amplification. The sequence of PCR products in the P1/P2 junction of the HAV genome revealed that the virus associated with the outbreak belongs to HAV subgenotype IA. HAV RNA was detected in ELISA negative specimens and in the specimen from a patient 2 days after the onset of symptoms using PCR amplification by CTAB method. These results indicate that PCR amplification was useful for the early diagnosis of hepatitis A infection.     

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.     

p53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation

BACKGROUND: The complications associated with anterior craniofacial resections for benign and malignant tumors were reviewed in 104 patients treated between January 1981 and June 1996. METHODS: Information regarding patient characteristics, histologic type, history of prior therapy, extent of the disease, extent of surgical procedure, and type of reconstruction were entered in a microcomputer database. To better understand and stage postoperative complications, we divided them into early (<14 days) and late (>14 days) according to the time of presentation, into major and minor depending on the morbidity potential of complication, and into local and systemic ones. Comparison between risk factors associated with complications was made using chi-square analysis with Yates' correction for continuity. Survival analysis was performed using the Kaplan-Meier product limit method. RESULTS: There were 8 (7.6%) postoperative deaths, with only 1 occurring from systemic complications. Complications occurred in 53 (48.6%) patients. Local major complications occurred in 49 (45%) patients, local minor in 29 (26.6%), and systemic in 11 (10%). Early complications occurred in 40 (38.5%) patients and late complications in 13 (12.5%) patients. These complications developed during a period ranging from 1 day to 5 months. More than one complication occurred in a number of patients. Bacterial contamination leading to local septic complications was the principal cause of morbidity, accounting for 54.7% (29/53) of complications. Major complications included meningitis in 8 patients associated with cerebrospinal fluid leak in 7, cerebral abscess in 2, sepsis in 1, and subdural hemorrhage in 1, all of which resulted in death except for one case. The extent of the craniofacial resection (p = .011) was the most important factor associated with major complications. Invasion of the dura and the type of reconstruction of the anterior skull base were the most important factors related to cerebrospinal fluid leakage (p = .048 and p = .032) and meningitis (p = .011). CONCLUSION: Contemporary surgical approaches and methods of reconstruction have enabled skull base surgeons to extend their cranial base resections and increase the 5-year survival rates of patients. Nevertheless, significant complications persist. Knowledge and high index of suspicion together with early recognition of these complications are essential for effective management of patients undergoing craniofacial resection. The factors related to major complications found in this study stressed the need to develop more effective methods to prevent contamination of intracranial structures.     

Functional adrenal cortical tumors in childhood: a study of ploidy, p53-protein and nucleolar organizer regions (AgNORs) as prognostic markers

The Washington State Patrol Crash Database and computerized hospitalization records for 1989-1993 were used to determine total hospital charges billed for motor vehicle collision injuries to drivers whose crash reports contained any indication of alcohol use. In this population-based study, total hospital charges were summed, and mean charges and lengths of stay were computed within alcohol use and insurance coverage status categories in an attempt to evaluate the hospital charges billed to public funding and private insurance. Of the total hospital charges for drivers with injuries from motor vehicle collisions for which a police-reported indicator of alcohol use status was available, 43% (U.S.$64.8 million) were for drivers who reportedly had been drinking. At the time of discharge, Medicaid was identified as the payor for 47% of these hospitalizations. The mean hospital charge billed per collision was greater for drinking (U.S.$18,258) than nondrinking drivers (U.S.$14,181). Drinking drivers also had longer hospital stays, even after adjustment for patient age, gender and injury severity. During this time in Washington state, the average annual amount billed at discharge for initial inpatient care of injuries to drivers who reportedly had been drinking at the time of the motor vehicle collision was U.S.$13 million. This includes only the amount assessed by the hospital at the time of discharge for treatment of the initial injury and does not include other related medical charges for rehabilitation or outpatient care, or for doctors' or laboratory fees. As increasing pressures of managed and capitated care lead to a shift of financial risk from the federal government and insurers to states and providers, the financial burden of specific, potentially preventable conditions such as this will receive greater attention.     

Quantitative analysis of cellular proliferative activity in 35 T-cell non-Hodgkin's lymphomas. Use of proliferating cell nuclear antigen and Ki-67 (MIB-1) antibodies and nucleolar organizer regions

OBJECTIVE: To analyze-cellular proliferation in non-Hodgkin's lymphoma (NHL) of T-cell origin using three variables available on histologic paraffin, dewaxed sections. STUDY DESIGN: The study group consisted of 35 T NHLs (22 low and 13 high grade). Two immunohistochemical methods established the percentage of cells expressing proliferating cellular nuclear antigen (PCNA), or PCNA index, and the Ki-67 antigen, or MIB-1 index. The third method quantified nucleolar organizer regions (NORs) with an image analyzer, giving the NOR area and number of NORs; an internal reference on lymphocytes was used. RESULTS: For the PCNA index, each subtype of low grade NHL demonstrated a difference as compared with high grade NHL except for angioimmunoblastic type NHL (AILD). The difference between the two grades became significant after including AILD-type NHL within high grade NHL (P = .02). The MIB-1 index gave similar results. The PCNA index and MIB-1 correlated (r = .55, P = .008). The relative NOR area and number of NORs differed significantly between the two grades (P < 10(-4) and P < 10(-2); the absolute NOR area differed to a lesser degree (P = .02), and no difference was observed for the absolute number of NORs (P = .07), stressing the importance of an internal reference. NOR areas and numbers correlated highly (r = .90 for relative and .78 for absolute variables, P < 10(-4)). No relation was found between PCNA and MIB-1 indexes. CONCLUSION: Correlations between these variables and grades of malignancy, between the two indices with each other and between the AgNOR variables with each other, including referring to internal lymphocytes, were reported for these T NHL-like tumors in studies on B NHL. The proliferative character of the AILD-type T NHL was in accordance with their worse prognosis. The absence of a correlation between PCNA or MIB-1 indices and NOR variables may reflect a biologic difference between B and T NHLs in a shorter cell cycle or more important functional activity in T NHL.     

Clinical relevance of p53 index and expression of proliferating cell nuclear antigen and Ki-67 in gastric cancer

To improve the therapeutic outcome for inoperable non-small-cell lung cancer, we applied definitive thoracic radiotherapy combined with concurrent administration of carboplatin and etoposide. We retrospectively analyzed 55 eligible patients with Stage III disease. The one-year rate of overall survival (OAS) and distant metastasis-free survival (DMFS) of the total group were 46.1% and 36.1%, respectively. Twenty-nine patients developed thoracic failures (52.7%) and 23 (41.8%) distant failures. Using univariate and multivariate analyses, radiation dose, performance status and LDH were revealed as significant prognostic factors of OAS, and LDH had a strong adverse effect on DMFS. Leucopenia of Grade 3 or higher was noted in 75.9%, anemia in 55.6%, thrombocytopenia in 59.3%, esophagitis in 20.4%, and lung injury in 10.9%. Sufficient gain was not obtained by our strategy, and higher morbidity, especially of lung, was noted than was expected. It was suspected that simultaneous use of oral etoposide might increase radiation pneumonitis, so one should take special care of unexpected toxicity in concurrent chemoradiotherapy. Both the hyperfractionated technique of radiotherapy and the time-dose modification of anti-tumor drugs should be considered in further steps.     

Assessment of the expression of p53, MIB-1 (Ki-67 antigen), and argyrophilic nucleolar organizer regions in carcinoma of the extrahepatic bile duct

Group B streptococci (GBS) are an important cause of neonatal sepsis, pneumonia and meningitis. In the early phase of infection, macrophages and polymorphonuclear cells (PMN) are the first immune cells that interact with GBS. In this in vitro study, to gain insight into GBS-macrophage interaction in the absence of type-specific antibodies, we examined the features of GBS survival in thioglycollate-elicited murine peritoneal macrophages and the effect of GBS on the protein kinase C (PKC)-dependent transduction pathway. Our results demonstrate that type Ia GBS, strain 090 (GBS-Ia) and type III GBS strain COH 31r/s (GBS-III), after in vitro phagocytosis survive and persist intracellularly in macrophages for up to 24 and 48 hr, respectively. However, macrophage activation by interferon-gamma (IFN-gamma) and lipopolysaccharide from Escherichia coli (LPS) caused a significant reduction in the time of intracellular persistence. Macrophage activation by IFN-gamma and LPS seems to be a multifactorial event involving multiple intracellular signal pathways also including PKC. Since PKC is one of the components in the signal network leading to macrophage activation and an important target for several intracellular micro-organisms, we wondered whether PKC could have a role in intracellular GBS survival. Both PKC depletion by treatment with phorbol 12-myristate 13-acetate (PMA) for 18 hr and PKC inhibition by Calphostin C rendered macrophages more permissive for the intracellular GBS survival. Furthermore, GBS-infected macrophages were unable to respond to PMA and LPS, activators of PKC, by inducing antimicrobial activity. The ability of GBS to impair PKC-dependent cell signalling was also demonstrated by the reduced c-fos gene expression in GBS-infected macrophages with respect to control macrophages, after LPS stimulation. In conclusion, our results indicate that GBS survive in macrophages and impairment of PKC signal transduction contributes to their intracellular survival.     

Apoptosis, bcl-2 protein, and Fas antigen in thymic epithelial tumors

We examined 35 thymic epithelial tumors by immunohistochemical techniques to investigate the extent of apoptosis detected by in situ end-labeling of fragmented DNA and the expression of bcl-2 and Fas. There were 22 thymomas (4 medullary, 9 mixed, 3 predominantly cortical, 6 cortical), 3 well-differentiated thymic carcinomas (WDTCs), and 10 high-grade thymic carcinomas (HGTCs), according to the Müller-Hermelink histologic classification. The HGTC showed the highest apoptotic index of the tumor cells. The apoptotic indices of the thymomas and WDTCs were significantly lower than those of the HGTCs (P < .001), but there was no significant difference among each type of thymoma and the WDTCs. bcl-2 protein was expressed in the tumor cells of the medullary-type thymomas and of the HGTCs but not in the other types of thymoma or WDTC. Fas antigen was negative in the HGTCs but was predominantly expressed in thymomas and WDTCs with advanced clinical stages (P < .05). From the immunostaining pattern, WDTC might be more likely to belong to the class of thymoma than to the category of HGTC.     

Detection of human papillomavirus DNA sequences in oral squamous cell carcinomas and their relation to p53 and proliferating cell nuclear antigen expression

BACKGROUND: The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. METHODS: Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. RESULTS: Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Human papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-16 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. CONCLUSIONS: These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes.     

Number of nucleoli and nucleolar organizer regions per nucleus and nucleolus--prognostic value in canine mammary tumors

The carbohydrate residues of the surface coat of 20 axenic cultures of Blastocystis hominis were studied using FITC-labelled lectins (ConA, WGA, DBA, HPA, SBA, PNA, UEAI and LPA). The specific affinity of reactive lectins was determinated by competitive inhibition assay with specific carbohydrates or by enzymatic pre-treatment of cells. All stocks strongly bound ConA and HPA; WGA, UEAI and LPA were partially reactive, and the remaining lectins were nonreactive. Inhibition assays showed abolition (WGA, LPA, UEAI and HPA) or partial reduction (ConA) of lectin affinity, which demonstrated the specificity of binding assay. These results indicate that B. hominis has surface components containing alpha-D-mannose, alpha-D-glucose, N-acetyl-alpha-D-glucosamine, alpha-L-fucose, chitin and sialic acid.     

Prognostic implications of proliferating cell nuclear antigen (PCNA), AgNORs and P53 in non-Hodgkin's lymphomas

We investigated the prognostic value of proliferating cell nuclear antigen (PCNA) and p53 oncoprotein expression and of nucleolar organiser region (NOR) scoring, in relation to classic clinicopathological parameters, in a series of non-Hodgkin's lymphomas (NHL). Paraffin embedded tissue from 91 patients with NHL was stained immunohistochemically with the monoclonal antibodies PC-10 (PCNA) and DO-1 (p53) and histochemically with the AgNOR technique. The median follow-up was 48 (4 to 193) months. The impact of PCNA and p53 expression and of AgNOR number on survival was tested using univariate as well as multivariate analysis, in order to circumvent the heterogeneity in histologic grade, type and therapy. Univariate analysis identified seven variables related to overall survival: histologic type and grade, clinical stage, chemotherapy, p53 labelling index (LI), PCNA LI and AgNOR score, whereas only one parameter i.e. histologic grade influenced disease-free survival. In multivariate analysis stage, PCNA LI and AgNOR score predicted independently overall survival. PCNA was also the only independent predictor of post-relapse survival and histologic grade the most important indicator of disease-free survival. In conclusion, PCNA expression and AgNOR number may be better predictors of overall and post-relapse survival than histologic grade. The latter remains the most valuable prognostic indicator of disease-free survival.     

p53 and c-jun expression in urinary bladder transitional cell carcinoma: correlation with proliferating cell nuclear antigen (PCNA) histological grade and clinical stage

OBJECTIVE: To investigate p53 and c-jun oncoproteins and proliferating cell nuclear antigen (PCNA) in transitional cell urinary bladder carcinomas (TCCs) and to determine their relationships to tumour grade, stage and survival. MATERIALS AND METHODS: The expression of p53, c-jun and PCNA was studied using immunohistochemistry in formalin-fixed, paraffin-embedded tissues in a series of 110 TCCs. RESULTS: 58% of our cases were positive for p53 and 88% for c-jun. A statistically very significant correlation (p < 0.0001) was observed between p53 and c-jun (r = 0.781), p53 and PCNA (r = 0.772), c-jun and PCNA (r = 0.831) as well as between each of the two oncoproteins and the histological grade and clinical stage (p < 0.001). There was no correlation of either p53, PCNA or c-jun with clinical outcome in terms of patients survival. CONCLUSION: p53 and c-jun proteins' overexpression are strongly related to rapid tumour cell proliferation and hence with aggressive growth in urinary bladder TCC. PCNA score remains an important prognostic index in transitional cell carcinoma of the bladder.     

Improvements in the silver-staining technique for nucleolar organizer regions (AgNOR)

The silver-staining technique for nucleolar organizer regions (AgNOR) of Ploton et al., as popularized by Crocker, is being widely used for evaluation of nucleolar function, especially in neoplasia. It suffers from limited reliability, background staining, precipitates, and fading of the sections. Factors were identified that affect these problems. The oxidation-reduction level and gelatin used are particularly important. An improved procedure is presented which incorporates pre-reduction of the sections, selection of an optimal gelatin, and post-treatment of the sections to produce a permanent preparation. It is compatible with many fixatives and with other stains used before or after the silver stain.     

Proliferative potential of meningiomas evaluated by proliferating cell nuclear antigen expression

Meningiomas are principally benign in nature. Some meningiomas, however, grow fast or recur even after total removal. The biological behavior of meningiomas often can not be predicted from conventional histopathological studies. A monoclonal antibody against proliferating cell nuclear antigen (PCNA) was used to investigate the usefulness of the PCNA index as a parameter to estimate the proliferative activity of meningiomas. Fifty-two meningiomas were examined. The mean PCNA index of recurrent meningiomas (3.37 +/- 0.92%) was significantly higher than that of non-recurrent meningiomas (1.12 +/- 0.51%) (p < 0.005). The PCNA indices of recurrent cases were all higher than 2.0%. A semilog linear regression analysis between tumor doubling time and PCNA index showed a significant correlation (r = 0.90, p < 0.05). An inverse linear correlation between PCNA index and interval to recurrence was observed (r = 0.62, p < 0.05). A good linear correlation was also shown between PCNA index and BUdR labeling index (r = 0.88, p < 0.01). The results of this study suggest that, providing the methods of tissue processing, immunostaining and counting of positive nuclei are unified, the PCNA index is a useful parameter for estimating the biological behavior of meningiomas.     

Expression of proliferating cell nuclear antigen in corneas kept in long term culture

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.