Avidin-biotin Complex Technique Research Articles

Pattern of Molecular Phenotypes of Breast Carcinomas using Immunohistochemistry in a District Hospital in Nigeria

Background: Cancer of the breast is the most common organ-specific cancer, affecting women worldwide with disproportionately higher mortality seen among women of Africa ancestry. Molecular phenotyping is crucial for tailored treatment of such patients. Aim: The aim of the study is to describe the molecular phenotypes of carcinomas of the breast based on their oestrogen, progesterone, and HER-2/Neu receptors status in our centre. Design: This was a retrospective analysis of 136 histologically diagnosed breast carcinomas, for which oestrogen, progesterone, and HER2 receptors status data were available at the Pathology Unit in a Nigerian District Hospital. Materials and Methods: Relevant biodata and pathology and molecular information of all patients with histologically diagnosed breast cancer (BC) were extracted from patients' forms and reports archived in the department. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue blocks of 136 (54.4%) cases of the BCs utilizing the avidin-biotin complex technique. Results: The ages of the patients ranged from 17 to 74 years, and the mean age was 47.3 ± 11.7 at diagnosis. All affected patients were females except for a male teenager. oestrogen, progesterone, and HER-2/Neu receptor-positive breast carcinomas were 43.4% (59), 39% (53), and 27.2% (37), respectively. Triple-negative breast carcinoma (39%, 53/136) was the most prevalent phenotype, followed by Luminal A (33.8%, 46/136) and Luminal B (14.7%, 20/136), while HER2-enriched (12.5%, 17/136) was the least common molecular subtype in our series. Conclusion: Breast carcinomas are predominantly of the triple-negative molecular phenotype in our setting and commonly affected young women.

Cultural and immunohistochemical evaluation of bacterial agents in caprine pneumonia in Nigeria

Goats are important protein-producing animals but pneumonia, in spite of the huge investments in small ruminant production, remains a major limitation. This study evaluates the prevalence of important bacterial pathogens associated with caprine pneumonia in Nigeria using cultural and immunohistochemical techniques. One hundred and fifty goat lungs were randomly examined from macroscopic, cultural isolation and microscopic changes using standard techniques. The common bacteria identified were formalin inactivated for bacterine and polyclonal antibody production. Immunohistochemical staining was performed against the bacteria antigens using the avidin-biotin peroxidase complex technique. The data were presented in frequencies and percentages, and compared using non-parametric statistics at α = 0.05. The pathology in the caprine lungs included broncho-interstitial pneumonia (41), interstitial pneumonia (27), and bronchopneumonia (72). Ten of the lungs were normal. Bacterial isolation yielded Mannheimia haemolytica (40%), Pasteurella multocida (20%), Escherichia coli (13%), Klebsiella pneumoniae (10%), Streptococcus sp. (8%), Baccillus sp. (5%), and Pseudomonas sp. (3%). Mannheimia haemolytica (Mh) and Pasturella multocida (Pm) were the most common isolated bacteria. The bacterial antigens were detected on the surface of respiratory bronchiolar, and alveolar epithelia, within inflammatory exudate and in cytoplasm of alveolar macrophages. One hundred twenty (80%) of the caprine lungs were positive for the Mh and Pm antigens; 47 for Mh, 59 for Pm, 14 for both, and 30 negative for either antigens. Significant differences were observed in the staining intensities of the antigens. This study validated Mannheimia haemolytica and Pasteurella multocida as most important bacterial pathogens in caprine pneumonia in Nigeria. Thus, adequate control of caprine pneumonia should consider bacterine or multivalent vaccines incorporating these important bacteria to curb morbidity and mortality of goats.

Partial Oxygen Pressure Affects the Expression of Prognostic Biomarkers HIF-1 Alpha, Ki67, and CK20 in the Microenvironment of Colorectal Cancer Tissue.

Hypoxia is prognostically important in colorectal cancer (CRC) therapy. Partial oxygen pressure (pO2) is an important parameter of hypoxia. The correlation between pO2 levels and expression levels of prognostic biomarkers was measured in CRC tissues. Human CRC tissues were collected and pO2 levels were measured by OxyLite. Three methods for tissue fixation were compared, including formalin, Finefix, and Finefix-plus-microwave. Immunohistochemistry (IHC) staining was conducted by using the avidin-biotin complex technique for detecting the antibodies to hypoxia inducible factor-1 (HIF-1) alpha, cytokeratin 20 (CK20), and cell proliferation factor Ki67. The levels of pO2 were negatively associated with the size of CRC tissues. Finefix-plus-microwave fixation has the potential to replace formalin. Additionally, microwave treatment improved Finefix performance in tissue fixation and protein preservation. The percentage of positive cells and gray values of HIF-1 alpha, CK20, and Ki67 were associated with CRC development (P < 0.05). The levels of pO2 were positively related with the gray values of Ki67 and negatively related with the values of HIF-1 alpha and CK20 (P < 0.05). Thus, the levels of microenvironmental pO2 affect the expression of predictive biomarkers HIF-1 alpha, CK20, and Ki67 in the development of CRC tissues.

Immunohistochemical localisation and effect of matrix metalloproteinases and their inhibitors on canine spontaneous periodontitis

Periodontitis is commonly observed in dogs. In human medicine, it is well documented that matrix metalloproteinases (MMPs) are involved in the destruction of the periodontium. Therefore, the aim of this...

Manual immunohistochemistry staining of mouse tissues using the avidin-biotin complex (ABC) technique.

There are many variations on the immunohistochemistry (IHC) procedure, but all are based on attachment of a primary antibody to a unique epitope on or within the cell. This step is followed by incubation of the cell/primary antibody complex with another, secondary antibody that recognizes the species in which the primary antibody was produced. The secondary antibody has an indicator molecule attached to it. The indicator produces a colored reaction product at the site of original epitope, allowing visualization. This basic two-antibody "sandwich" procedure has many modifications that include other layers of antibodies and numerous indicators, but all variations depend upon the unique ability of antibodies to recognize specific epitopes or antigenic determinants. The procedure described here is called the ABC (avidin-biotin complex) technique. The method utilizes the high avidity of biotin for avidin, which allows formation of a strong bond. The reagents described in this technique produce a gold/brown reaction product that identifies the epitope of interest.

Arrangement of sympathetic fibers within the human common peroneal nerve: implications for microneurography

Recently, interest has grown in the firing patterns of individual or multiunit action potential firing patterns in human muscle sympathetic nerve recordings using microneurography. Little is known, however, about sympathetic fiber distribution in human lower limb nerves that will affect the multiunit recordings. Therefore, the purpose of this study was to examine the sympathetic fiber distribution within the human common peroneal nerve using immunohistochemical techniques (tyrosine hydroxylase, avidin-biotin complex technique). Five-micrometer transverse and 10-μm longitudinal sections, fixed in formaldehyde, were obtained from the peroneal nerve that had been harvested from three human cadavers (83 ± 11 yr) within 24 h of death. Samples of rat adrenal gland and brain served as controls. Sympathetic fiber arrangement varied between left and right nerves of the same donor, and between donors. However, in general, sympathetic fibers were dispersed throughout ∼25-38 fascicles of the peroneal nerve. The fibers were grouped in bundles of ∼2-44 axons or expressed individually throughout the fascicles, and the distribution was skewed toward smaller bundles with median and interquartile ratio values of 5 and 1 axons/bundle, respectively. These findings confirm the bundled organization of sympathetic axons within the peroneal nerve and provide the anatomical basis for outcomes in microneurographic studies.

Prognostic Value of Disseminated Tumor Cells in the Bone Marrow of Patients with Operable Primary Breast Cancer: A Long-term Follow-up Study

Detection of disseminated tumor cells (DTC) in primary breast cancer (BC) patients' bone marrow (BM) seems to be a surrogate marker of tumor spread and an independent prognostic factor for disease-free and overall survival. Here we present the largest single-center cohort of patients (n=1378) with the longest observation time (median 82.0months). Immunocytochemical staining was performed using murine monoclonal antibody 2E11 with the avidin-biotin complex technique. At primary surgery, 49% of patients showed MUC-1 positive cells inside their BM. Patients without BM DTC had significantly more often T1-tumors (P=0.007) with less often affected axillary lymph nodes (P<0.001). We observed a significantly higher incidence of distant metastases in DTC positive patients (P<0.001). This leads to a reduced disease-free survival (P<0.0001). Furthermore, in DTC positive patients there was a higher mortality rate and, accordingly, a reduced overall survival (P<0.0001). Due to the presence of BM DTC, patients with a clinically poorer outcome can be identified at primary surgery. We therefore suggest that DTC analysis can be used as a prognostic factor and monitoring tool in clinical trials. Future study concepts relating to DTC should aim at identification of BC patients who may profit from adjuvant systemic therapy.

A preliminary study on the arrangement of sympathetic fibres within the human common fibular nerve

Recently, interest has grown in the firing patterns of individual or multi-unit action potentials (AP) in human muscle sympathetic nerve recordings using microneurography. Little is known, however, about sympathetic fibre distribution in human lower limb nerves that will affect the multi-unit recordings. Therefore, the purpose of this study was to examine the sympathetic fibre distribution within the human common fibular nerve using immunohistochemical techniques (tyrosine hydroxylase, avidin–biotin complex technique).

Universal Markers of Thyroid Malignancies: Galectin-3, HBME-1, and Cytokeratin-19

Difficulties in diagnosis of thyroid lesions, even with histologic analysis, are well known. This study has been carried on to evaluate the role of immunohistochemical markers including galectin-3, Hector Battifora mesothelial cell-1 (HBME-1), and cytokeratin-19 in the diagnosis and differential diagnosis of benign and malignant thyroid lesions. The expressions of galectin-3, HBME-1, and cytokeratin-19 were tested in formalin-fixed, paraffin-embedded tissues from 458 surgically resected thyroid lesions including non-neoplastic and neoplastic lesions. Immunostaining with standard avidin-biotin complex technique was performed by using monoclonal antibodies. In malignant neoplastic thyroid lesions, galectin-3, HBME-1, and cytokeratin-19 were diffusely expressed in general. Diffuse expression rates of these three markers were 72.3% (47/65), 70.7% (46/65), and 76.9% (50/65), respectively. The use of galectin-3, HBME-1, and cytokeratin-19 may provide significant contributions in the differential diagnosis of malignant thyroid tumors. Although focal galectin-3, HBME-1, and cytokeratin-19 expression may be encountered in benign lesions, diffuse positive reactions for these three markers are characteristic of malignant lesions. It has concluded that cytokeratin-19 alone and its combinations with other markers were more sensitive in accurate diagnosis of papillary carcinoma than the other combinations; meanwhile, there were similar results for follicular carcinomas with HBME-1 alone and its combinations.

Targeting Inflammatory Demyelinating Lesions to Sites of Wallerian Degeneration

In Theiler’s murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelinating lesions, and the distribution of axonal damage present during the early phase of infection corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal damage recruits inflammatory cells to sites of Wallerian degeneration, leading to demyelination. Three weeks after TMEV infection, axonal degeneration was induced in the posterior funiculus of mice by injecting the toxic lectin Ricinus communis agglutinin (RCA) I into the sciatic nerve. Neuropathology was examined 1 week after lectin injection. Control mice, infected with TMEV but receiving no RCA I, had inflammatory demyelinating lesions in the anterior/lateral funiculi. Other control mice that received RCA I alone did not develop inflammatory lesions. In contrast, RCA I injection into TMEV-infected mice induced lesions in the posterior funiculus in addition to the anterior/lateral funiculi. We found no differences in lymphoproliferative responses or antibody titers against TMEV among the groups. This suggests that axonal degeneration contributes to the recruitment of inflammatory cells into the central nervous system by altering the local microenvironment. In this scenario, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model).

Nerve Regeneration after Radiofrequency Application

Background Many patients with chronic tendinosis have experienced early pain relief after application of bipolar radiofrequency treatment. It is hypothesized that the mechanism of action may be the acute degeneration and/or ablation of sensory nerve fibers. Hypothesis After ablation or degeneration by bipolar radiofrequency, nerve fibers will have the ability to regenerate with time. Study Design Controlled laboratory study. Methods Eighteen Sprague-Dawley rats were used in this study. These rats were divided into 3 groups (30, 60, and 90 days after bipolar radiofrequency). These rats were treated with 2 points of bipolar radiofrequency applications to the left hindpaws with the Topaz microdebrider device. Right hindpaws were used as the contralateral control. Tissues were processed for neural class III β-tubulin or calcitonin gene-related peptide immunohistochemistry by using the free-floating avidin biotin complex technique. The numbers of neural class III β-tubulin—immunoreactive and calcitonin gene-related peptide-immunoreactive nerve fibers in the epidermis were counted and compared with those in the contralateral control. Results Although the numbers of nerve fibers demonstrated by both the antibodies of neural class III β-tubulin and calcitonin gene-related peptide were significantly decreased (P < .0001) until 60 days after bipolar radiofrequency treatment, regeneration of the epidermal nerve fibers occurred 90 days after treatment. Conclusion Bipolar radiofrequency treatment induced degeneration of sensory nerve fibers immediately after treatment, but by 90 days posttreatment, there was evidence of complete regeneration. Clinical Relevance Early degeneration followed by later regeneration of nerve fibers after bipolar radiofrequency treatment may explain long-term postoperative pain relief after microtenotomy for tendinosis.

Human B-lymphocytes Express α2-6-Sialylated 6-Sulfo-N-acetyllactosamine Serving as a Preferred Ligand for CD22/Siglec-2

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize alpha2-6-sialylated glycan as a specific ligand. Here we propose that the alpha2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the alpha2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The alpha2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the alpha2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as alpha2-6-sialylation may regulate CD22/Siglec-2 functions in humans.

Pain Relief through an Antinociceptive Effect after Radiofrequency Application

Background Many patients with chronic tendinosis have experienced early pain relief after application of bipolar radiofrequency treatment. Pathologic nerve ingrowth or nerve irritation in the tendon has been considered as a possible cause of the pain experienced with tendinosis. Hypothesis Bipolar radiofrequency treatment will ablate nerve fibers, resulting in pain relief. Study Design Controlled laboratory study. Methods Eighteen Sprague-Dawley rats were used in this study. Eight rats were treated with 2 points of bipolar radiofrequency applications applied to the hind paws with the Topaz microdebrider device, 6 sham rats had a needle applied to the hind paws, and there were 4 control rats. Tissues were processed for neural class III β-tubulin (TUJ-1) or calcitonin gene-related peptide (CGRP) immunohistochemistry by using the free-floating avidin-biotin complex technique. The numbers of TUJ1-immunoreactive and CGRP-immunoreactive nerve fibers in the epidermis were counted and compared with sham and control. Results The number of nerve fibers demonstrated by both the antibodies of TUJ1 and CGRP were significantly decreased (P = .0002-.002) during the first 2 weeks after bipolar radiofrequency treatment. Macroscopically, the foot pad showed 2 dimples on the surface after bipolar radiofrequency treatment. Although it still showed a scar after 7 days, after 14 days it looked no different than the untreated contralateral control foot pad and foot pad of the sham group. Conclusion Bipolar radiofrequency treatment induced acute degeneration and/or ablation of sensory nerve fibers. Clinical Relevance Degeneration or ablation of nerve fibers after bipolar radiofrequency treatment may explain the early postoperative pain relief after microtenotomy for tendinosis.

A rabbit model of cervical spondylosis established by stimulation of wind, cold and dampness

To make an animal model of cervical spondylosis (arthralgia syndrome type) with stimulation of wind, cold, and dampness. Twenty-four 8 months old male New Zealand white rabbits were randomly allocated into four groups: normal control group, light stimulation group, moderate stimulation group and severe stimulation group. The wind speed was 10.8-13.8 m/s, the temperature was (5+/-0.5)degrees centigrade, and the humidity was 100%. The rabbits of light, moderate, and severe stimulation groups were kept in the above-mentioned environments for 4 hours everyday, and for a total of 32, 64, and 128 hours, respectively. The intervertebral discs were stained with HE method, and observed with a light microscope. Prostaglandin E(2) (PGE(2)), 6-ketone-prostaglandin F1alpha (6-K-PGF(1alpha)) and thromboxane B(2) (TXB(2)) contents were measured by ELISA. Fas and Bcl-2 expressions were examined by immunohistochemical avidin-biotin peroxidose complex technique. Interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta) mRNA expressions were examined by reverse transcription-polymerase chain reaction. The nucleus pulposus of rabbits in the light and moderate stimulation groups shrunken, and in the severe stimulation group, the anulus fibrosus loosed or ruptured, and the cartilage end-plate became proliferated. Compared with rabbits in the normal control group, the PGE(2) content rose in the light stimulation group, the contents of PGE(2), 6-K-PGF(1alpha), and TXB(2) increased, the expressions of IL-1beta and TNF-alpha mRNAs and Fas were up-regulated, and the expressions of TGF-beta mRNA and Bcl-2 were down-regulated in the moderate and severe stimulation groups. The expression of Fas was up-regulated mostly and Bcl-2 was down-regulated mostly in the severe group. Moderate and severe stimulations of wind, cold and dampness can lead to degeneration of cervical intervertebral discs of rabbits. The model corresponds to the theory of traditional Chinese medicine about arthralgia syndrome caused by wind, cold and dampness.

Immunohistochemical screening for viral agents in cheetahs (Acinonyx jubatus) with myelopathy

Numerous cases of acute-onset progressive ataxia, hindlimb paresis and paralysis of unknown aetiology occurred during 1993 to 2003 in cheetahs (Acinonyx jubatus) within the European Endangered Species Programme (eep). This...

Immunohistochemical Localization of Sperm‐Preserving Proteins in the Ram Reproductive Tract

Previously, we reported that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury, and that 2 proteins of approximately 14 (P14) and 20 (P20) kDa, the main components of fraction 6 isolated by exclusion chromatography, are responsible for this protective effect. The objective of the present study was to localize P14 and P20 in tissues of the ram reproductive tract to determine their origin. Antiserum generated against purified P14 and P20 reacted with proteins in seminal vesicles and vas deferens by Western blot analyses of protein tissue extracts. However, these antisera failed to detect P14 and P20 in testis, prostate, efferent ductules, bulbourethral glands, and epididymis (caput, corpus, and cauda). Immunohistochemical analyses by both indirect immunofluorescence and the avidin-biotin complex technique confirmed that only seminal vesicles showed reactivity, restricted to the secretory cells, with both antibodies. Obtained results indicate that P14 and P20 are secreted specifically in the seminal vesicles. To further confirm that P14 and P20 are specifically expressed in seminal vesicles, we used Northern blot analyses to investigate the expression of both proteins in seminal vesicles and vas deferens. These assays corroborated again that P14 and P20 were specifically expressed in seminal vesicles. Consequently, we suggest referring to these 2 proteins as RSVP14 and RSVP20, respectively, according to their origin and molecular weight.

Immunohistochemical localization of inhibin and activin subunits, activin receptors, and Smads in ovarian clear cell adenocarcinoma

Our purpose was to examine the immunolocalization of inhibin, activin and the activin signaling system in ovarian clear cell adenocarcinoma tissue. Tissue samples of ovarian clear cell adenocarcinoma were collected from ten women. The patients' ages ranged from 41 to 71 years (mean +/- standard deviation; 53.4+/-9.9). Their surgicopathological staging was based on the criteria of the International Federation of Gynecology and Obstetrics (FIGO); there were 5 patients with stage I, 3 with stage II, and 2 with stage III. We examined the immunohistochemical localization of inhibin/activin alpha-subunit, betaA-subunit, activin A, and activin receptor types IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 using an avidin-biotin peroxidase complex technique. With the antibodies against the betaA-subunit, and activin A, we observed positive immunoreactive staining in the cytoplasm of clear cell adenocarcinoma, whereas we did not observe any staining for the alpha-subunit of inhibin. We observed positive immunoreactive staining in the cell cytoplasm and surface with the antibodies against the activin receptors, types IA, IB, IIA and IIB. We observed positive immunoreactive staining in the cytoplasm and nucleus with the antibodies against the Smad2, Smad3 and Smad4. It is suggested that activin A, not inhibins, might be secreted by ovarian clear cell adenocarcinoma, and that an activin signal transduction pathway might exist in ovarian clear cell adenocarcinoma.

Prostate-specific membrane antigen is undetectable in choroidal neovascular membrane

BackgroundChoroidal neovascular membrane (CNVM) is one of the leading causes of severe visual loss and is often associated with age-related macular degeneration (AMD). Various modalities of treatment, including photocoagulation and surgery, are being considered as options, but with limited success. Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein expressed in benign and malignant prostatic tissues, in some non-prostatic tissues, and in the endothelium of tumor-associated neovasculature of non-prostatic neoplasm. Some studies have suggested that the expression of PSMA is restricted to endothelium from tumor-associated neovasculature and might be stimulated by some tumor-secreted angiogenic factors. However, no previous study demonstrating PSMA expression in non-related tumor neovasculature, such as CNVM, has been performed to date. Furthermore, demonstration of PSMA expression in CNVM in AMD patients could reveal a novel target for antineovascular therapy. The purpose of this study was to evaluate the immunohistochemical expression of PSMA in CNVM from AMD.MethodsImmunohistochemical analysis, with a standard avidin-biotin complex technique, was performed using an anti-PSMA mouse monoclonal antibody in 30 specimens of surgically excised CNVM from AMD patients. Antibody to an endothelial cell specific marker, factor VIII, was used to confirm the location of the endothelial cells.ResultsThe angiogenic microvessels of the 30 cases demonstrated negative staining to PSMA while factor VIII was expressed in all cases. Seventy-five percent of the secretory-acinar epithelium of the prostatic hyperplasia specimen stained positive, confirming that the immunohistochemical technique was correctly performed.ConclusionThe absence of PSMA expression in non-tumoral neovasculature supports the theory, previously suggested, that endothelial cell PSMA expression may be stimulated by one or more tumor-secreted angiogenic factors. Angiogenesis is very important in neoplasia and the endothelial expression of PSMA in tumor-associated neovasculature may represent a target for antineovasculature-based therapy. The absence of PSMA expression in CNVM suggests that PSMA may not be a potential target for antineovasculature-based therapy.

Dissecting the Axoneme Interactome: The Mammalian Orthologue of Chlamydomonas PF6 Interacts with Sperm-Associated Antigen 6, The Mammalian Orthologue of Chlamydomonas PF16

Dissecting the Axoneme Interactome: The Mammalian Orthologue of Chlamydomonas PF6 Interacts with Sperm-Associated Antigen 6, The Mammalian Orthologue of Chlamydomonas PF16

Intracellular localization of NS3 and C proteins in chronic hepatitis C

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. The cytoplasmic localization of both proteins dominated over nuclear localization. The total amount of NS3 protein exceeded that of C protein. At the ultrastructural level, we observed both proteins in endoplasmic reticulum membranes and mitochondria, but small amount of both proteins was seen also in cell nuclei. The amount of either protein did not correlate with liver histology in our patients. No correlation could be disclosed between content of both proteins and HCV RNA levels in serum. Content of NS3 demonstrated negative correlation with activity of alanine transaminase. In conclusion, the new aspect in present studies involved localization of C and NS3 proteins at the levels of light and electron microscopy in children. For the first time also intramitochondrial localization of NS3 protein was demonstrated.