George K. Koukoulis

Extracellular matrix proteins and their receptors in the normal, hyperplastic and neoplastic breast.

We studied by immunohistochemistry, the distribution of tenascin (Ten), cellular fibronectin (cFn), laminin and certain pertinent extracellular matrix protein receptors in normal human female breast, variants of fibrocystic disease (FCD), benign tumors, and ductal and lobular carcinomas. Monoclonal antibodies (mAb) to Ten, extradomain A containing cFn (EDAcFn), A and B chains of laminin, and beta-1 (beta-1) and different alpha subunits of intergrins were used. In in-situ ductal and lobular carcinomas, laminin staining had focal gaps, Ten-immunoreactivity displayed periductal or periacinar bands, and cFn showed broad and intense periductal staining; strong reactions for beta-1 and alpha-6 were noted in the basal cytoplasm of non-neoplastic myoepithelial cells while few tumor cells stained weakly. In infiltrating ductal and lobular carcinomas (IDC, ILC), laminin reactivity was weak, uneven or absent around neoplastic clusters whereas stromal staining for Ten and cFn was extensive and strong. In most IDC, moderate beta-1 and alpha-6 staining involved variable subpopulations; one mucinous carcinoma stained strongly and diffusely. In 20-40% of cells in ILC, beta-1 and alpha-6 were localized in delicate, ramified cytoplasmic processes. Indirect immunofluorescence studies with mAbs to other alpha-integrin subunits suggest that in various breast carcinomas only alpha-3 is expressed in tumor cells and that the vessels contained alpha-1 integrin. As compared with the normal breast, FCD and benign tumors, reactivity for Ten and cFn is increased in breast carcinomas while laminin is attenuated and decreased or absent; yet, Ten cannot be regarded as a carcinoma marker since it can be detected in benign tumors, FCD, and even in the normal breast.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimitochondrial antibodies of immunoglobulin G3 subclass are associated with a more severe disease course in primary biliary cirrhosis

Background/Aims: Primary biliary cirrhosis (PBC) is characterised by the presence of immunoglobulin (Ig) G antimitochondrial antibodies (AMA), which are routinely detected by indirect immunofluorescence (IFL) using composite rodent tissue substrate. The IgG subclass distribution and clinical significance of IFL‐detected AMA in patients with PBC have not been previously studied in detail.

Cysteinyl Leukotriene Receptors Are Expressed by Tonsillar T Cells of Children With Obstructive Sleep Apnea

BACKGROUND Increased expression of cysteinyl leukotriene receptors (cysteinyl leukotriene receptor-1 [LT1-R]; cysteinyl leukotriene receptor-2 [LT2-R]) has been detected in adenotonsillar tissue from children with sleep-disordered breathing (SDB) compared to control subjects. LT1-R has been localized in myeloperoxidase-positive cells. This phenomenon possibly contributes to lymphoid tissue enlargement and may be related to systemic inflammation. OBJECTIVE To characterize cells expressing LT1-R and LT2-R in tonsillar tissue and assess serum C-reactive protein (CRP) levels in children with and without SDB. METHODS Immunohistochemistry with LT1-R and LT2-R antibodies was used to examine tonsils from children who had tonsillectomy (with or without adenoidectomy) for SDB and from control subjects operated for recurrent tonsillitis/otitis. All participants underwent preoperative polysomnography and measurement of morning serum CRP. RESULTS Fifteen children with SDB (mean age +/- SD, 6.4 +/- 2.1 years; apnea-hypopnea index, 9.6 +/- 5.6 episodes per hour) and 11 control subjects (age, 7.5 +/- 2.8 years; apnea-hypopnea index, 7 +/- 0.3/h) were examined. Immunoreactivity for LT1-R and LT2-R was detected in tonsillar extrafollicular areas of all subjects with SDB but not of control subjects. Cells expressing leukotriene receptors were CD3+ lymphocytes. Children with SDB and control subjects were similar regarding CRP levels: 0.11 +/- 0.15 mg/dL vs 0.09 +/- 0.15 mg/dL, respectively (p > 0.05). CONCLUSIONS Tonsils of children with SDB but not of control subjects have enhanced expression of cysteinyl leukotriene receptors in T lymphocytes without an associated increase in serum CRP concentration. Up-regulation of LT1-R and LT2-R could potentially promote tonsillar enlargement in children with obstructive sleep apnea.

Immunolocalization of integrins in the normal lung and in pulmonary carcinomas.

Cryosections of normal adult lung (n = 7) and pulmonary epithelial tumors, including squamous (n = 8), adeno (n = 8), bronchioloalveolar (n = 5), and large cell (n = 4) carcinomas (SCC, ACC, BAC, LCC), carcinoids (Cd, n = 7), and neuroendocrine carcinomas (NEC) of variable grades (n = 14) were immunostained by the avidin-biotin peroxidase (ABC) method with monoclonal antibodies to the alpha1-6 and alpha(v) and the beta1-4 integrin subunits. Normal adult alveolar septae showed variably intense immunoreactivity for alpha1,3,6 and beta1, whereas reactions for alpha5 and alpha(v) were weaker and uneven; the remaining integrin subunits were not detected. Bronchial and bronchiolar epithelium showed variably intense staining for alpha2.3,6,v and beta1,4. Reactions were often, though not invariably, basally polarized. SCC, ADC, and LCC showed variably intense reactions for alpha2.3,6,v and beta1,4. BAC were strongly and uniformly stained for alpha1.3 and beta1. In Cd, alpha1,2,3,v and beta1 reactions were noted, whereas in NEC, weak alpha1,3 and beta1 staining was detected with only traces of alpha6 and alpha(v). We conclude that alveolar epithelial cells do not express the hemidesmosome-associated, laminin-binding integrin alpha6beta4 of the bronchial epithelium but rather the alpha1beta1 and alpha3beta1, collagen IV, and laminin receptors, respectively. SCC, ADC, and sampled LCC express an integrin repertory qualitatively similar to that of the bronchial epithelium. Distinct from the latter, the integrin repertory of BAC parallels that of the alveolar epithelium by its strong expression of the multipotential alpha1beta1 and alpha3beta1 integrins. NEC tumors do not display the laminin receptors alpha6beta4 and alpha6beta1 shown by SCC and ADC but express instead alpha1beta1, a collagen IV-laminin receptor rarely found in epithelial neoplasms except for BAC. In NEC tumors, integrins, especially alpha2, decrease with dedifferentiation. Notably distinct from epithelial mesotheliomas, the major fibronectin-binding integrin alpha5beta1 was not found in any type of lung carcinoma.

High-risk human papillomavirus (HPV) in parotid lesions

Although several studies have reported that oropharyngeal infection with HPV may predispose to tumorigenesis, little is known about the etiological factors of salivary gland tumors and the presence of HPV. We studied 9 parotid lesions for HPV infection including an oncocytoma, an acinic cell carcinoma, a high-grade adenocarcinoma, a low-grade polymorphous adenocarcinoma, a Warthins tumor and 2 pleomorphic adenomas, a lymphoepithelial cyst and a lipoma of the parotid gland. DNA was extracted from formalin-fixed and paraffin-embedded tissue sections. Solution PCR for HPV detection was performed using the GP5+/GP6+ primers, while HPV typing was carried out by multiplex PCR for HPV6, 11, 16, 18, and 33; positive samples were recorfirmed by PCR with specific primers for each type. Quantitative real-time PCR for the high-risk HPV genotypes 16, 18, 31, 33, 35, 52, 58 and 67 was also performed to quantitate the viral load. Finally, in situ PCR was employed with HPV16-specific primers by direct-detection method. Seven of the 9 parotid lesions were HPV positive while 6 of these 7 had been infected by HPV16 and/or HPV18 oncogenic types. High viral load of highrisk genotypes of HPV was found in the oncocytoma, in one of the pleomorphic adenomas, and in the Warthins tumor. Finally, in situ PCR indicated that HPV16 amplification occurred in the salivary gland tumors. This is the first time that highrisk HPV genotypes are detected in these histological types of parotid lesions, suggesting the possible involvement of the virus in the disease.

Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.

Autoimmune hepatitis (AIH) is an unresolving progressive liver disease of unknown etiology characterized by hypergammaglobulinemia, autoantibodies detection and interface hepatitis. Due to the absence of specific diagnostic markers and the large heterogeneity of its clinical, laboratory and histological features, AIH diagnosis may be potentially difficult. Therefore, in this in-depth review we summarize the substantial progress on etiopathogenesis, clinical, serological and histological phenotypes of AIH. AIH has a global distribution affecting any age, both sexes and all ethnic groups. Clinical manifestations vary from asymptomatic to severe or rarely fulminant hepatitis. Hypergammaglobulinemia with selective elevation of IgG is found in most cases. Autoimmune attack is perpetuated, possibly via molecular mimicry, and favored by the impaired control of T-regulatory cells. Histology (interface hepatitis, emperipolesis and hepatic rosette formation) and autoantibodies detection although not pathognomonic, are still the hallmark for a timely diagnosis. AIH remains a major diagnostic challenge. AIH should be considered in every case in the absence of viral, metabolic, genetic and toxic etiology of chronic or acute hepatitis. Laboratory personnel, hepato-pathologists and clinicians need to become more familiar with disease expressions and the interpretation of liver histology and autoimmune serology to derive maximum benefit for the patient.

Clinical significance of organ- and non-organ-specific autoantibodies on the response to anti-viral treatment of patients with chronic hepatitis C.

Background  Development of organ‐ and non‐organ‐specific autoantibodies has been reported in hepatitis C virus patients treated with interferon‐α plus/minus ribavirin.

Immunoloealization of integrins in the normal and neoplastic colonic epithelium

SummaryCryosections of normal colon (NC), tubular and villous adenomas (TA, VA), and variably differentiated colon adenocarcinomas (CA) were immunostained with monoclonal antibodies to α1−6 and αv, and β1−4 integrin subunits; select samples were stained for cytokeratin (Ck) 20 and villin. In NC, α2 staining was strongest in crypt cells; α1,3 and αv, and β1,3 and β4, and Ck 20 and villin predominated in superficial enterocytes. In TA and VA, monolayered glands showed integrin, Ck 20 and villin patterns that differed slightly from both crypt and superficial enterocytes. Complex glands in VA showed decreased integrin staining and basal polarization; Ck 20 and villin were strong only in luminal cells. CA showed overall weaker integrin staining than adenomas. Regardless of invasion depth, well formed malignant glands mimicked TA; pleomorphic glands mimicked VA with focal basal integrin polarization and solid clusters displayed scanty integrins, uneven Ck 20, and villin in occasional cells. Diverse integrins in crypt compared with superficial enterocytes reflect changing adhesive requirements as cells migrate and terminally differentiate. Decreasing expression and altered distribution of integrins, Ck 20 and villin noted in TA, VA, and in CA of increasing grade indicate that certain adhesive and cytoskeletal features more closely relate to glandular architecture than to depth of invasion.

Superoxide dismutase activity in children with chronic liver diseases

BACKGROUND/AIMS Liver disease in infancy has multiple etiologies. As reactive oxygen intermediates are involved in several types of tissue damage, we have investigated whether different forms of liver disease in infancy are associated with increased free radical generation, using an indirect approach in which superoxide dismutase (a free radical scavenger) activity is determined in the liver tissue. METHODS A total of 48 liver biopsies performed at diagnosis were evaluated retrospectively. Nine infants had biliary atresia, eight Alagille syndrome, seven alantitrypsin deficiency and 12 cryptogenic hepatitis. As controls we studied 12 biopsies with normal histology obtained from seven children with portal vein thrombosis and five children who underwent biopsy for management reason but had no liver disease. Superoxide dismutase activity in liver biopsy specimens was measured using the cytochrome C method by spectrophotometry and expressed as U SOD/mg protein. RESULTS Superoxide dismutase activity was significantly increased in biliary atresia (1.25 +/- 0.56 U SOD/mg protein, p<0.0001) and Alagille syndrome (1.31 +/- 0.56 U SOD/mg protein, p<0.0001) as compared with al-antitrypsin deficiency (0.75 +/- 0.3 U SOD/mg protein), neonatal hepatitis (0.72 +/- 0.37 U. SOD/mg protein) and normal controls (0.4 +/- 0.7 U. SOD/mg protein). The highest level of SOD activity was found, however, in control children with portal vein thrombosis (2.09 +/- 0.96 U SOD/mg protein; p<0.0001 as compared to the other groups). CONCLUSION Superoxide dismutase, a key enzyme in free radical protection, is increased significantly in the liver tissue of infants with cholestatic liver disease due to bile duct damage and in children with portal vein thrombosis, suggesting that products of free radical reactions are involved in the pathogenesis of these disorders.

Habib EndoHPB: A Novel Endobiliary Radiofrequency Ablation Device. An Experimental Study

ABSTRACT Background: The Habib EndoHPB is a bipolar radiofrequency (RF) catheter developed to be introduced across malignant strictures of the bile ducts, so that RF energy can locally ablate the tumor prior to stent placement. This experiment aims to assess the ability of the catheter to coagulate the wall of the common bile duct (CBD) in a porcine model, to establish power requirement and time parameters and correlate them to the depth of thermal injury, and to assess the ease of operation of the device. Methods: The CBD was catheterized using the device in 20 pigs. RF energy was applied to the CBD wall with various generator settings. The pigs were sacrificed 24 hr after the application and the CBD was excised for histological analysis. Results: The device was easy to handle. Statistically significant correlations between the power, the time of RF application, and the thermal injury depth were found. Conclusion: The Habib EndoHPB catheter can effectively deliver RF energy intraluminally in the porcine CBD. Clinical studies are warranted in order to define proper settings for safe and efficient use in malignant biliary obstruction.