Angiotensin II: immunohistochemical study in Sardinian pterygium.

The angiotensin II (Ang II) is the principal effector peptide of the RAS system. It has a pleiotropic effect and, beside its physiological role, it has the property to stimulate angiogenesis and activate multiple signalling pathways related to cell proliferation. The purpose of the study was to determinate the Ang II expression and localization in Sardinian pterygium and normal conjunctiva by immunohistochemistry, and its possible involvement in the development and progression of the disease. Twenty-three pterygiums and eleven normal conjunctiva specimens obtained from Sardinian patients, were processed for paraffin embedding and assessed for the immunohistochemical revelation of Ang II. Significant Ang II expression was identified in pterygium and conjuntica. Particularly, thirteen pterygium specimens (n=13) displayed exclusively moderate to strong nuclear staining; some specimens (n=5) showed exclusively a moderate cytoplasmatic immunoreactivity, and few specimens (n=2) displayed moderate to strong immunoreactivity in both cytoplasm and nucleus. Statistical significance difference in respect of nuclear and cytoplasmatic localization was observed between normal conjunctiva and pterygium (P=0.038).The results showed a predominant intranuclear localization of Ang II in pterygium epithelial cells, in spite of conjunctiva that mainly showed cytoplasmatic localization. In view of these results, we hypothesized a possible gene expression modulator role played by Ang II in pterygium.

Nestin expression associates with poor prognosis and triple negative phenotype in locally advanced (T4) breast cancer.

Nestin, an intermediate filament protein, has traditionally been noted for its importance as a neural stem cell marker. However, in recent years, expression of nestin has shown to be associated with general proliferation of progenitor cell populations within neoplasms. There is no reported study addressing nestin expression in T4 breast cancer patients. Thus, the aim of the present study was to investigate, through immunohistochemistry, the expression and distribution of nestin in T4 breast cancer, in order to determine its association with clinical and pathological parameters as well as with patients' outcome. Nestin was detectable in tumoral cells and in endothelial cells of blood microvessels, and it is significantly expressed in triple-negative and in inflammatory breast cancer (IBC) subgroups of T4 breast tumours. The Kaplan-Meier analysis showed that the presence of nestin in tumoral cells significantly predicted poor prognosis at 5-years survival (P=0.02) and with borderline significance at 10-years of survival (P=0.05) in T4 breast cancer patients. On the basis of these observations, we speculate that nestin expression may characterize tumours with an aggressive clinical behavior, suggesting that the presence of nestin in tumoral cells and vessels may be considered an important factor that leads to a poor prognosis. Further studies are awaited to define the biological role of nestin in the etiology of these subgroups of breast cancers.

Expression of survivin protein in pterygium and relationship with oxidative DNA damage.

Ultraviolet radiation is known to cause oxidative DNA damage and is thought to be a major factor implicated in the pathogenesis of pterygium. Among all the photo-oxidative DNA products, the 8-hydroxydeoxyguanosine (8-OHdG) is regarded a sensitive and stable biomarker for evaluating the degree of DNA damage. The protein p53 is a major cell stress regulator that acts to integrate signals from a wide range of cellular stresses. UV radiation has a carcinogenic effect resulting in DNA damaged cells with loss of normal growth control. This assumption is supported by the association between UV-B exposure and activation of survivin, a member of the inhibitor of apoptosis protein family (IAP), highly up-regulated in almost all types of human malignancy. In this study we demonstrate, for the first time in pterygium, the immunohistochemical presence of survivin, and investigate the correlation between survivin, p53 and 8-OHdG. Our results demonstrate that oxidative stress could lead to a significant activation of survivin expression, suggesting that this might be an important event in the development of pterygium, inducing and supporting a hyperproliferative condition. Survivin expression in pterygium would counteract UV-B-induced apoptosis and would cooperate with loss of p53. The co-operation between survivin and functional loss of p53 might provide a general mechanism for aberrant inhibition of apoptosis that could be responsible for the development of pterygium and its possible progression to neoplasia.

Nuclear survivin is associated with disease recurrence and poor survival in patients with cutaneous malignant melanoma.

AIMS: Survivin is expressed in neoplastic cells and appears to be associated with resistance to therapy and shorter survival in various types of tumours. The aim of the present study was to determine whether nuclear or cytoplasmic expression of survivin is related to disease recurrence and overall survival of patients with Stage I and II melanoma according to the American Joint Committee on Cancer (AJCC) staging system. METHODS AND RESULTS: Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections of primary cutaneous melanoma from 50 patients. Survival rates were estimated using the Kaplan-Meier method and compared using the log rank test. Association of clinical variables (gender, age, tumour location, thickness, Clark level and AJCC stage) with survivin expression was analysed by Fisher's exact test. Patients with nuclear immunoreactivity for survivin had an increased risk of disease recurrence during the first three postoperative years (P < 0.05) and of death (P < 0.05). Cytoplasmic immunoreactivity was not correlated with either survival or clinical variables. CONCLUSIONS: Nuclear presence of survivin may be an independent biomarker for disease recurrence and overall survival in patients with Stage I and II melanoma.

Prognostic prediction of the immunohistochemical expression of p16 and p53 in cutaneous melanoma: a comparison of two populations from different geographical regions.

p16INK4a and p53 are tumor-suppressor genes frequently altered in various malignancies, including cutaneous melanoma. The purpose of the study was to establish the prognostic value of immunohistochemical expression of p16INK4a a and p53 in sporadic cutaneous melanoma (CM) in two regions with a high-risk for melanoma in Italy and Ecuador. Immunohistochemical staining of p16 and p53 was performed in samples of primary CM from 82 patients with Stage I and II melanoma according to the American Joint Committee on Cancer (AJCC) staging system. Survival differences between categories of p16 or p53 expression were analyzed using the product-limit procedure (Kaplan-Meier method, log-rank test). Clinical variables (gender, age, tumor location, Clark's level, thickness) were correlated with survival and p16 or p53 expression. p16 nuclear immunoreactivity was observed in 85% of Italian patients compared to 48.7% of Ecuadorians; a small number of cases showed p53 immunoreactivity in both populations. Only nuclear p16 expression exhibited a significant correlation with survival (Italians p=0.001, Ecuadorians p=0.017) but did not appear to correlate with any clinicopathological parameter. No significant difference was observed in survival with regard to p53 expression or cytoplasmic p16. Our results demonstrate that nuclear expression of p16 can be considered a molecular prognostic factor in patients with sporadic CM and indicate its importance as a clinical marker.

Finding of conjunctival melanocytic pigmented lesions within pterygium.

AIMS: Conjunctival pigmented lesions have characteristic clinical and histopathological appearances. Melanocytic pigmented lesions commonly occur in the conjunctiva, although they have not been previously reported in pterygium, a common lesion which originates from conjunctiva. Our aim was to evaluate the possibility of an association between pterygium and conjunctival melanocytic pigmented lesions. METHODS AND RESULTS: A total of 80 samples of pterygium excised from Ecuadorian patients in 2002 were collected. Clinical data were available regarding age, sex, race and place of residence. Histological sections were evaluated for the presence of melanocytic pigmented lesions. Nine cases of conjunctival melanocytic, pigmented lesions within pterygium were found and were classified according to the histopathological criteria previously published for pigmented lesions of the conjunctiva, as naevi and primary acquired melanosis (PAM) with varying degrees of atypia. Five of the nine cases showed primary acquired melanosis without atypia, while two cases had atypia; one case showed features of compound naevus and one lesion was designated as subepithelial naevus. CONCLUSIONS: Our findings suggest that conjunctival melanocytic, pigmented lesions occasionally occur in pterygium. All surgically removed pterygia should undergo careful histopathological examination.

Detection of human papillomavirus DNA in pterygia from different geographical regions.

BACKGROUND/AIMS: The aetiology and pathogenesis of pterygia remain unclear and the involvement of human papillomavirus (HPV) is controversial. 41 pterygia from two geographic locations were evaluated for the presence of HPV DNA. METHODS: 41 pterygium biopsies (17 from Italy and 24 from Ecuador) were analysed using the L1C1 and PU-1ML primer sets by polymerase chain reaction (PCR) and DNA sequence analysis. RESULTS: 22 of the 41 pterygia (54%) were positive for HPV, including all 17 Italian cases and 5/24 (21%) Ecuadorean cases. DNA sequencing of the 22 positive cases showed that 11 were HPV type 52, four were type 54, five were candHPV90, and two of unknown genotype. CONCLUSIONS: The major differences in the frequency of HPV in geographically distant populations might suggest a possible explanation for the vast differences in the reported detection rates. Three subtypes of HPV were found in this sample of pterygia. None the less, these results suggest that HPV may have a pathogenic role in pterygium.

Immunohistochemical study of human pterygium.

The purpose of this study has been to evaluate the immunohistochemical characteristics of human pterygial tissues in order to ascertain the possible contribution of an immunological mechanism in the pathogenesis of pterygium and to investigate the presence in the pterygial tissues of some melanoma-associated antigens, in order to evaluate if there may be a small possibility of correlation of the two diseases. Human biopsy specimens of pterygium were obtained by surgery for pterygium excision. Tissue segments were fixed and processed for paraffin embedding. Microtome sections were treated for the immunohistochemical demonstration of IgA, IgM, IgG, CD3, CD20, CD68, HLA-DR, Protein S100, HMB45, and Melan A using the avidin-biotin peroxidase method or the streptavidin biotin-alkaline phosphatase method. The findings suggest that all the effector components of the mucosal immune system are present in the human pterygium and, among the most sensitive markers for melanoma, only S100 shows immunoreactivity. An immunopathogenetic mechanism seems to be responsible for the pathogenesis of pterygium, perhaps being caused by pre-existing conjunctivitis or microtrauma in combination with the patient's predisposition. No correlation between pterygium and melanoma was found.

Subcellular localization of epidermal growth factor in human submandibular gland.

Epidermal growth factor in human submandibular gland was localized at the subcellular level by means of an immunogold staining method. Labelling was observed in serous acini and ducts. In the acini, gold particles were found within secretory granules, indicating that the growth factor is released into the saliva through granule exocytosis. In the ductal system, the most intense reactivity was revealed in the principal cells of striated ducts. In these cells, an abundant population of small cytoplasmic vesicles was specifically stained. Immunoreactive vesicles were found both apically and basally, suggesting that ductal cells can release their products not only into the saliva but also into the interstitium.

The presence of a local immune system in the upper blind and lower part of the human nasolacrimal duct.

The nasolacrimal duct is exposed to exogenous agents, including potentially harmful microorganisms, coming from the eye surface by the lacrimal sac, and from the nasal cavity by the inferior meatus of the nose. The upper blind and lower part of the human nasolacrimal duct were examined immunohistochemically to ascertain the presence and localization of immunoglobulin-producing cells and the epithelial expression of IgA, IgM, and IgG in order to verify the possible antimicrobial properties of this duct. IgA-, IgM-, and IgG-positive immunocompetent cells were recognizable in the lamina propria of the upper blind and lower part of the human nasolacrimal duct, while an evident immunoreactivity for sIgA, IgM, and IgG was demonstrated in the cytoplasm of the apical epithelial cells. The results suggest that all the effector components of the mucosal immune system are present in that area of the human nasal mucosa next to the opening of the nasolacrimal duct as well as in the human lacrimal sac.

Immunohistochemical localization of secretory immunoglobulins in the main excretory duct of the human submandibular gland.

The localization of IgA, IgG, and IgM was investigated immunohistochemically in the mucosal surface of the main excretory duct of the human submandibular gland in order to verify the possible antimicrobial properties of this duct. Only secretory IgA-immunoreactivity was recognized in the epithelial cells of the duct. An intense immunoreactivity was observed in the cytoplasm of some cells and at the luminal surface of most of the cells. Clusters of IgA-positive immuno-competent cells were also recognizable in the subepithelial layers. No reactivity for IgG and IgM was noticed. The results suggest that the ductal epithelium may actively be involved in the release of secretory IgA, which could play a prominent role in the local defense mechanism of the duct.

Immunoglobulins in the human male genitourinary tract.

Human male genitourinary tract was processed for the immunohistochemical demonstration of IgG and IgM, in order to verify the presence and the source of these immunoglobulins. Only the epithelia of the prostate gland and urethra showed a marked IgM-immunopositivity, localized in the cytoplasm of apical cells. An intense immunoreactivity for IgG was observed in the apical cells of the vesical epithelium; a less intense immunoreactivity for IgG in the pelvic apical epithelial cells was found. Immunostaining in the ureteric epithelium was absent. Clusters of IgM- and IgG-positive immunocompetent cells were present in the subepithelial layers of the organs observed in our study. This study indicates that IgM, present in mucosal surfaces, derived from an active secretion, as well as secretory IgA, whereas most of the IgG present in the human seminal plasma were probably due to transudation process. Immunoreactivity for IgG in the vesical and pelvic epithelia could depend upon to virus-specific IgG antibodies produced by the mucosa. This in turn can contribute to the total antiviral activity in mucosal secretions.

Local immune response in the skin of the external auditory meatus: an immunohistochemical study.

Cerumen plays an important role in the protection of the external auditory meatus against several kinds of damage. Its hydrophobic properties, due to the high concentration of lipids, shelter the canal from physical damages, while other components probably protect against certain microbial strains. Nevertheless there has been considerable dispute in the literature with regard to the antibacterial activity of cerumen. Because of the importance of the role of immunoglobulins (Ig) in local defense mechanisms, we attempted to study, by immunohistochemical methods, the presence and localization of the cells necessary to activate an Ig-mediated immune response and the epithelial expression of immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) in order to obtain information about a local immune response in those areas of the skin that take part in cerumen production. Our findings indicate that in the human skin of the external auditory canal the cells necessary to activate an antibody-mediated immune response were localized in the different layers of the epidermis and/or in the dermis surrounding the sebaceous and ceruminous glands and the piliary follicle, while an intense immunoreactivity for IgA and IgG was observed in the epithelial layers of the skin. The results suggest that the external auditory canal is protected from the insults of pathogens by an antibody-mediated local immune response, because all the effector components of an active local immune system are present.

IgM and IgG in the human male genital tract.

In human seminal plasma it is possible to measure the levels of IgG and IgA, while IgM has been detected in trace amounts in only a few subjects. The source of these antibodies is unknown, although, under certain conditions, they seem to be produced in the sex tissues. Male human genital tract was processed for the immunohistochemical demonstration of IgG and IgM to verify the presence and the source of these antibodies. Only the epithelia of the prostate gland and urethra showed positivity to the immunohistochemical reaction, indicating the presence of IgM-secreting epithelial cells. Clusters of IgM- and IgG-positive immunocompetent cells were also observed in the subepithelial layers of all organs studied. The findings suggest that IgM are present in mucosal surfaces as a result of active secretion, as well as secretory IgA, whereas most of the IgG present in the human seminal plasma are probably derived by transudation process.

Histochemical demonstration of acetylcholinesterase activity in human Meibomian glands.

The distribution of acetylcholinesterase activity in human Meibomian glands was evaluated using enzyme-histochemical methods. The butyrylcholinesterase (BuChE) inhibitor, tetraisopropyl pyrophosphoramide (iso-OMPA), was used to localize acetylcholinesterase (AChE) activity, the AChE inhibitor, 1,5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51), was used to localize BuChE activity, and eserine was used to inhibit all cholinesterase activity in control incubations; the appropriate specific inhibitors for competing enzymic activities were added to the incubation medium. At the light microscopic level, acetylcholinesterase reaction product appeared as cytoplasmic brown deposits, often crystalline. A very dense accumulation of AChE-positive nerve fibers was seen in the form of a network around the acinar and the ductal tissue of the glands. No discrete nerve endings were observed, whereas a strong reaction was elicited in some fibers in close association with blood vessels. These observations suggest that the cholinergic system is involved in the regulation of the Meibomian glands secretory function.

A histochemical and immunohistochemical study of certain defense mechanisms in the human lacrimal sac epithelium.

The mucosal surface of the human lacrimal sac represents an area exposed to exogenous agents including potentially harmful microorganisms. The human lacrimal sac was examined histochemically to identify glycoproteins, and immunohistochemically to identify secretory IgA. Neutral and acid glycoconjugates were detected mainly in the cytoplasm of the surface cells of the columnar stratified epithelial lining. The same reactions were recognized in occasional clusters of secretory cells forming intraepithelial glands in the lining of the lacrimal sac. The presence of secretory IgA in the cytoplasm of the apical epithelial cells was demonstrated. The results indicate that the lacrimal sac mucosa possesses certain active defense mechanisms against ascending infections.

Immunohistochemical study of secretory IgA in the human male reproductive tract.

Male human genital tract was treated for the immunohistochemical demonstration of secretory IgA in order to verify its own possible antimicrobial properties. An intensely positive immunoreaction for sIgA in the epithelial cells of prostate and urethral glands was observed; in the same organs the presence of immunoreactive cell clusters in the subepithelial layers was noted. Immunostaining in the epithelia of deferent duct, seminal vesicle, deferential ampulla, ejaculatory duct and bulbourethral glands was absent. The findings suggest that a local immune response sIgA-mediated is present in the lower male genital tract.

Immunohistochemical demonstration of secretory IgA in human urothelium.

The mucosal surface of the human urothelium represents a very large exposure area to exogenous agents, including potentially harmful microorganisms. Male human urothelium was treated for the immunohistochemical demonstration of secretory IgA (sIgA) in order to verify its own possible antimicrobial properties. An intense immunoreactivity for sIgA was observed in the apical cells of the urethral and vesical epithelia. The ureteric epithelium, at the luminal surface, showed discontinuous areas of less dense or completely absent reaction product. A less intense immunoreactivity was observed in the pelvic apical epithelial cells. The results suggest that sIgA play a prominent role in the local defence mechanisms of the lower urinary tract against ascending infections, whereas in the upper urinary tract the immuno-specific local defences seem reduced.

Human male genitourinary tract: an immunohistochemical study of secretory IgA.

Male human genitourinary tract was treated for the immunohistochemical demonstration of secretory IgA (sIgA) in order to verify its own possible antimicrobial properties. An intense immunoreactivity for sIgA was observed in the apical cells of the urethral and vesical epithelia. The ureteric epithelium, at the luminal surface, showed discontinuous areas of less dense or completely absent reaction product. A less intense immunoreactivity in the pelvic apical epithelial cells was found; an intensely positive immunoreaction for sIgA in the epithelial cells of prostate and urethral glands was also observed. The results suggest that a local immune response sIgA-mediated is present in the lower male urogenital tract.

Human urethral epithelium: immunohistochemical demonstration of secretory IgA.

Male human urethral epithelium was treated for the immunohistochemical demonstration of secretory IgA in order to verify its own possible antimicrobial properties. An intense immunoreactivity for sIgA in the entire cytoplasm and at the luminal surface of the apical epithelial cells was observed. The results suggest that sIgA play a prominent role in the local defense mechanisms of the urethra. Such mucosal defense mechanisms may be of significance in the defense of the urinary and genital tract against ascending infections.