Wiesława Biczysko

Expression of Cytokines (TNF-α, IL-1α, and IL-2) in Chronic Hepatitis C: Comparative Hybridocytochemical and Immunocytochemical Study in Children and Adult Patients

Hepatitis C virus (HCV) is one of the principal causes of hepatitis, which in more than 80% of cases leads to chronic lesions in the liver and involvement of extrahepatic organs. It remains unknown why the infection so frequently turns chronic, independently of patient age. Using immunocytochemistry (IHC) and in situ hybridization (ISH) (both linked to the ImmunoMax technique) we examined cell sources of TNF-α, IL-1α, and IL-2 in control and HCV-infected children and adults. We demonstrated augmented expression of all the cytokines in HCV-infected patients compared to controls. No differences were detected in amounts of studied transcripts or cytokine proteins between biopsies taken from HCV-infected children and adults. Expression of TNF-α was localized mainly in liver sinusoidal cells (macrophages, endothelial cells). A high proportion of hepatocytes demonstrated expression of TNF-α, IL-1α, and IL-2. In both groups of patients, higher amounts of cytokine proteins than studied transcripts were demonstrated. The augmented expression of TNF-α, IL-1α, and IL-2 in liver with a similar proportion of involved cells (mainly hepatocytes) in children and in adults points to participation of the cytokines in the pathogenesis of chronic hepatitis C. The expression is insufficient to terminate the infection and may be linked with the comparably frequent chronic transformation of HCV infection noted in children and adults.

Intracellular localization of NS3 and C proteins in chronic hepatitis C.

Background: The cellular localization of hepatitis C virus (HCV) proteins in chronic hepatitis C remains a debatable issue. Aim of the studies included cellular and subcellular localization of two HCV proteins, NS3 and C protein, in liver biopsies from 20 children with chronic hepatitis C employing commercially available monoclonal antibodies and a semiquantitative technique of the proteins expression. In the studies advantage was taken of (Strept)avidin‐biotin peroxidase complex and ImmunoMax technique.

p21/Wafl/Cipl cellular expression in chronic long-lasting hepatitis C: correlation with HCV proteins (C, NS3, NS5A), other cell-cycle related proteins and selected clinical data.

Studies indicate that proteins of hepatitis C virus (HCV) disturb expression of cell-cycle-related proteins. A disturbed cell-cycle control is a hepatocellular carcinoma (HCC) risk factor in patients with HCV-related liver damage. The present study aimed to analyse the cellular expression of p21/Wafl/Cipl (p21) in long-lasting chronic hepatitis C (CH-C), its correlation with the key oncogenic HCV proteins (C, NS3, NS5A), other cell-cycle-related proteins (PCNA, Ki-67, cyclin D1, p53) and selected clinical data. Archival liver biopsies, obtained from patients with CH-C, normal livers, and hepatocellular carcinoma (HCC) specimens were analysed by immunocytochemistry and ImmunoMax technique. In CH-C overexpression of p21 protein was demonstrated. Positive correlations of p21 protein expression in CH-C involved age of the patients, grading, and liver steatosis. Moreover, expression of p21 correlated significantly with expression of p53 protein, of D1 cyclin and Ki-67. Although Ki-67 antigen was related to p21 expression, only Ki-67 expression proved to be directly related to liver staging. Expression of the NS3 protein, which prevailed in CH-C patients, manifested correlation with p21 expression, and that of cyclin D1. In presence of preserved potential for regeneration, overexpression of p21 indicates inhibition of cell cycle in hepatocytes, which probably plays a protective role for the chronically damaged cells. Out of the three HCV proteins only NS3 seems to affect control of p21 protein expression in in vivo infection. Nevertheless, the studies indicate that neither expression of p21 protein nor that of viral NS3 protein can serve as a marker of progression of CH-C to HCC in vivo.

Detection of DNA, mRNA and early antigen of the human cytomegalovirus using the immunomax technique in autopsy material of children with intrauterine infection.

Abstract. The present study focuses on the immunomax technique in association with the avidin-biotin-peroxidase complex (ABC) technique and a non-isotopic variation of in situ hybridisation (ISH) for optimal microscopical detection of human cytomegalovirus (HCMV). The studies were performed on an archival paraffin material originating from five children deceased due to intrauterine infection. The results of immunocytochemical and hybridocytochemical studies, with or without amplification using biotinylated tyramine, were compared with the routine histopathological results and results obtained using the polymerase chain reaction (PCR). Early antigen (EA)-HCMV was demonstrated in approximately twice as many cells as detected in the routine staining and also in cells that seemed morphologically intact. The hybridocytochemical studies confirmed the presence of HCMV DNA in cells that were positive in the immunocytochemical tests and, in addition (using the ISH-immunomax technique), in cell nuclei of intact myocardial myocytes. In general, fewer cells manifested the presence of HCMV mRNA than the presence of HCMV DNA. The immunomax technique was found to be more sensitive than the techniques of classical immunocytochemistry or of ISH. The former technique permitted the documentation of a higher number of HCMV replication sites than could be detected using the latter techniques. However, the clinical course of HCMV infection or the cause of death of the children was not directly related to the intensity of HCMV expression in tissues.

p53 immunocytochemistry and TP53 gene mutations in patients with chronic hepatitis C virus (HCV) infection.

Chronic infection with hepatitis C virus (HCV) is regarded as a risk factor for hepatocellular carcinoma (HCC), mostly in patients with liver cirrhosis. Present study aimed at evaluation of cellular expression of p53 protein, genetic TP53 changes in liver samples and anti-p53 in serum of patients with chronic hepatitis C virus infection. The expression of p53 protein were analysed by immunocytochemistry in liver biopsies from adult patients with chronic, long-lasting hepatitis C. In order to detect TP53 mutations, PCR/SSCP and sequencing were performed. Antibodies against p53 in serum were determined using enzyme immunoassay (ELISA).In two out of 14 examined patients TP53 point mutations were detected in the liver samples. In the first patient, a substitution of C to T was demonstrated in position 1 of the codon 250, resulting in substitution of proline by serine. The other patient carried a substitution of C to G in position 13274 of the intron 6. The patient carrying mutation in the codon 250 demonstrated morphological traits of liver cirrhosis and had high number of p53-immunoreactive cell nuclei in tissue. None of the patients manifested elevated titres of serum anti-p53. In the liver, significant positive correlations were disclosed between expression of p53 on one hand and grading and staging on the other. A negative correlation was disclosed between cellular expression of p53 and duration time of infection. In conclusions, genetic changes in TP53 can be detected also in non-neoplastic lesions linked to chronic HCV infection.

Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin

Oral and oropharyngeal cancers are characterized by relatively low 5- year survival rates due to many factors, including local recurrence. The identification of new molecular markers may serve for the estimation of prognosis and thus augment treatment decisions and affect therapy outcome. The aim of this study was to describe the morphological characteristics and the DNA methylation status of the CDKN2A,CDH1, ATM, FHIT and RAR- genes in the central and peripheral part of the tumor and the surgical margin and evaluate their prognostic significance. 53 patients with oral and oropharyngeal cancer were enrolled to the prospective study, and had been primarily treated surgically. Correlations between morphological data, hypermethylation status and clinicopathological data, as well as prognosis, were assessed. Nuclei polymorphism highly correlated with T stage (p < 0.0001), N stage (p < 0.046), and metastases to the lymph nodes pN (p < 0.004 ). Also, the number of cells in irregular mitosis correlated with T stage (p < 0.004), and highly with pN (p < 0.009). The significance of CDKN2A hypermethylation as a good prognostic factor was also established in the Kaplan-Meir test. The ultrastructural analysis showed that none of the examined tumors had homogenous texture and that resection margin specimens clean in HE stained tissue samples frequently contained single tumor cells or few cells in groups surrounded by connective tissue. This indicates the superiority of electron microscopy over standard histopathological analysis. Thus, a combination of such morphological examination with epigenetic parameters described herein could result in the discovery of promising new prognostic markers of the disease.

Rola badań immunohistochemicznych (białka p53, cykliny D1) w prognozowaniu raka gruczołowato-torbielowatego (carcinoma adenoides cysticum) ślinianek

Summary Introduction Adenoid cystic carcinoma of the salivary glands ( carcinoma adenoides cysticum ) is malignant epithelial tumor of rare occurrence. Tumor of this kind has among salivary glands tumors uncertain prognosis and unpredictable course. The aim of the study was to characterize the patient population and the immunohistochemical analyses (p53 protein, cyclin D1). Material and methods The examined group consisted of 30 patients with adenoid cystic carcinoma of the salivary glands. The expression of p53 protein and D1 cyclin in the tumor was evaluated and the correlation between these proteins and the organ and clinical grading was defined. Results The immunohistochemical studies showed in 70% the positive staining for p53 protein and 90% for cyclin D1. There was not statistically significant difference between the advanced grading of the organic and clinical adenoid cystic carcinoma.INTRODUCTION Adenoid cystic carcinoma of the salivary glands (carcinoma adenoides cysticum) is malignant epithelial tumor of rare occurrence. Tumor of this kind has among salivary glands tumors uncertain prognosis and unpredictable course. The aim of the study was to characterize the patient population and the immunohistochemical analyses (p53 protein, cyclin D1). MATERIAL AND METHODS The examined group consisted of 30 patients with adenoid cystic carcinoma of the salivary glands. The expression of p53 protein and D1 cyclin in the tumor was evaluated and the correlation between these proteins and the organ and clinical grading was defined. RESULTS The immunohistochemical studies showed in 70% the positive staining for p53 protein and 90% for cyclin D1. There was not statistically significant difference between the advanced grading of the organic and clinical adenoid cystic carcinoma.

67. Ocena ultrastrukturalna obwodowych części raka gruczołowato – torbielowatego (ACC) ślinianek

Carcinoma adenoides cysticum jest rakiem wystepującym w roznych postaciach: gruczolowatym, sitowatym oraz litym. Kliniczno-morfologiczna analiza wskazuje, ze carcinoma adenoides cysticum charakteryzuje sie znaczną miejscową tendencją do wznowy; naciekaniem naczyn i nerwow oraz odleglymi przerzutami do pluc i kości nawet w kilka lat po usunieciu guza pierwotnego. Cel pracy : Celem naszej pracy byla analiza ultrastrukturalna obwodowych partii guza ze szczegolnym zwroceniem uwagi na cechy charakterystyczne wystepujące w trzech podstawowych typach histologicznych. Material i metodyka Material tkankowy pochodzil od 46 chorych z rakiem gruczolowato-torbielowatym ślinianek operowanych w Klinice Otolaryngologii AM w Poznaniu. Tkanke po usunieciu utrwalano wg Karnowskiego, a nastepnie dzielono, do rutynowego badania H+E i mikroskopu elektronowego. Pozostalą cześc zabezpieczano w bloczkach parafinowych. Dodatkowo wykonano badanie immunohistochemiczne dla fibronektyny i lamininy wg metody ABC. W badanym materiale wyodrebniono nastepujące typy histologiczne: 16 sitowatych, 17 gruczolowatych oraz 13 litych. Wyniki Podczas oceny ultrastrukturalnej nie stwierdzono typowych hitoarchitektonicznych form dla obwodowych cześci utkania. Jedynie zaobserwowano tendencje do grupowania sie komorek wokol fibrylarnych struktur utkania, ktore zawierają proteoglikany, glikozaminoglikany oraz kolagen typu IV. W typie litym komorki bardzo czesto pozbawione są polaryzacji i polączen miedzykomorkowych. Fibronektyna pojawia sie zarowno w ob.-rebie cytoplazmy jak i we wszystkich strukturach guza. Laminina jedynie w ścianie naczyn oraz strukturach podstawowych zarowno w postaci sitowatej i litej. Przerzuty do obwodowych wezlow chlonnych zanotowano u 12 chorych (10 typ lity, 2 typ tabularny).