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.

Serum concentrations of IL-2, IL-6, IL-12 and TNF-α in patients with alopecia areata.

Alopecia areata (AA) has been represented as a restricted T cell-mediated autoimmune disease. Several studies have shown that cytokines may play an important role in its pathogenesis although many pathways exist. We investigated cytokine (IL-2, IL-6, IL-12, and TNFα) levels in peripheral blood mononuclear cell (PBMC) of 105 patients with different grade and duration of alopecia areata, to confirm that T-cell responses in AA is regulated via mechanisms of peripheral T-cell tolerance. We observed that IL-12 levels are higher for patients with bigger extensions and tend to increase according to the duration of the AA; TNFα instead, is more related to the gender of the patients and to the duration. Therefore abnormalities in cytokines production, showed by our results, may suggest that T-cell responses in AA scalp are closely regulated via mechanisms of peripheral T-cell tolerance and therefore confirm that this disease has an immuno-pathogenesis. Our aim is to shed light upon the complexity of AA underlying mechanisms and indicate pathways that may suggest future treatments.

Comparitive effectiveness of finasteride vs Serenoa repens in male androgenetic alopecia: a two-year study.

The objective of this open label study is to determine the effectiveness of Serenoa repens in treating male androgenetic alopecia (AGA), by comparing its results with finasteride. For this purpose, we enrolled 100 male patients with clinically diagnosed mild to moderate AGA. One group received Serenoa repens 320 mg every day for 24 months, while the other received finasteride 1 mg every day for the same period. In order to assess the efficacy of the treatments, a score index based on the comparison of the global photos taken at the beginning (T0) and at the end (T24) of the treatment, was used. The results showed that only 38% of patients treated with Serenoa repens had an increase in hair growth, while 68% of those treated with finasteride noted an improvement. Moreover finasteride was more effective for more than half of the patients (33 of 50, i.e. 66%), with level II and III alopecia. We can summarize our results by observing that Serenoa repens could lead to an improvement of androgenetic alopecia, while finasteride confirmed its efficacy. We also clinically observed, that finasteride acts in both the front area and the vertex, while Serenoa repens prevalently in the vertex. Obviously other studies will be necessary to clarify the mechanisms that cause the different responses of these two treatments.

Genetic association of HLA-DQB1 and HLA-DRB1 polymorphisms with alopecia areata in the Italian population.

BACKGROUND: Alopecia areata (AA) is a multifactorial disease characterized by hair loss especially from the scalp. As for other autoimmune conditions, the major histocompatibility complex (HLA) region is associated with AA susceptibility. OBJECTIVE: To provide evidence for the association of specific HLA-DQB1 and HLA-DRB1 alleles with AA in an Italian population, using a case-control approach. METHODS: We performed a case-control study to investigate whether HLA-DQB1 and -DRB1 alleles predispose to AA in the Italian population. HLA class II typing was performed in 85 patients with AA and 210 healthy controls from the same ethnic group. RESULTS: An increased frequency of DQB1*03, coding for DQ7 heterodimers, and a decreased rate of the DQB1*06 allele were observed in patients when compared with controls; the greatest and significant difference was in the group of cases with a more severe phenotype [AA>50% patients (more than 50% hair loss) vs. controls, P=4·5×10(-3) , P(c)=0·031, odds ratio (OR) 2·01, 95% confidence interval (CI) 1·22-3·31 and P=2·5×10(-3) , P(c)=0·017, OR 0·22, 95% CI 0·07-0·72, respectively]. DQB1*03, serologically related to DQ8 or coding for DQ9 molecules, was not associated with AA susceptibility. Out of all patients, 65·9% carried DQ7 heterodimers compared with 49·5% of the controls (P=7·3×10(-3) , OR 1·97, 95% CI 1·17-3·32) and DQ7 prevalence rose to 76·3% in patients with AA>50% (P=1·7×10(-3) , OR 3·28, 95% CI 1·48-7·27). No significant difference was found in the distribution of DRB1 variants or phenotypes among cases and controls. CONCLUSION: Our data show a correlation between the HLA-DQB1 locus and the occurrence of AA in Italy supporting DQB1*03(DQ7) as a predisposing allele for the disease and the relevance of the HLA genetic test in the clinical management of AA.

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.

A morphological and 13C NMR study of the extramandibular fat bodies of the striped dolphin (Stenella coeruleoalba).

The molecular and histological structure of the fat bodies covering externally the posterolateral region of the jaw of the striped dolphin (Stenella coeruleoalba) was investigated by means of morphological and nuclear magnetic resonance techniques. The analyses of samples belonging to adult and juvenile individuals were performed with the aim of seeking the presence of age-related differences. In our study, the level of isovalerate (iso5:0) in the extramandibular fat of the juvenile individuals is comparable with those of the adult counterparts; conversely, longer isobranched fatty acids were detected in lower quantities in the juveniles together with a higher degree of unsaturation. The morphologic analyses revealed that, in both adults and juveniles, this fatty tissue is similar to univacuolar adipose tissue. However, in the juveniles, a muscular component was present, whereas only in adult subjects, enlarged and irregularly shaped cavities may be seen within the adipose tissue. These cavities, structurally organized as veins, may regulate blood flow in response to changing water temperature and stabilize thermal gradient within the jaw lipids. These data suggest that the molecular components and the histological organization can indicate a maturation of the organ with age that probably may reflect different sound reception properties.

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.

A histological and NMR study of the melon of the striped dolphin (Stenella coeruleoalba).

The melon, the echolocation organ of the striped dolphin (Stenella coeruleoalba), was investigated by morphological and high-resolution (13)C nuclear magnetic resonance (NMR) methods, in order to characterize structure and composition gradient at the histological and molecular level. Morphological analysis showed that the lipidic components are organized in an adipose tissue; moreover, a clear muscular component was observed. Age-related structural differences also were noted. Furthermore, NMR yielded detailed information at a qualitative-quantitative level on the lipid components.

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.

The synthetic cannabinoid WIN55212-2 decreases the intraocular pressure in human glaucoma resistant to conventional therapies.

The search for new ocular hypotensive agents represents a frontier of current eye research because blindness due to optic neuropathy occurs insidiously in 10% of all patients affected by glaucoma. Cannabinoids have been proposed to lower intraocular pressure by either central or peripheral effects but a specific mechanism for this action has never been elucidated. We recently demonstrated the presence of the central cannabinoid receptor (CB(1)) mRNA and protein in the human ciliary body. In the present study we show that the synthetic CB(1) receptor agonist, WIN 55212--2, applied topically at doses of 25 or 50 microg (n = 8), decreases the intraocular pressure of human glaucoma resistant to conventional therapies within the first 30 min (15 +/- 0.5% and 23 +/- 0.9%, respectively). A maximal reduction of 20 +/- 0.7% and 31 +/- 0.6%, respectively, is reached in the first 60 min. These data confirm that CB(1) receptors have direct involvement in the regulation of human intraocular pressure, and suggest that, among various classes of promising antiglaucoma agents, synthetic CB(1) receptor agonists should deserve further research and clinical development.

The human eye expresses high levels of CB1 cannabinoid receptor mRNA and protein.

We used reverse transcriptase polymerase chain reaction to detect the expression of the central and peripheral cannabinoid receptors (CB1 and CB2, respectively) mRNA, and Western blotting to show the presence of the CB1 protein in subregions of the human eye. CB2 mRNA transcripts were undetectable, while levels of CB1 mRNA were significantly expressed in the human retina (25.8 +/- 2.46%), ciliary body (210 +/- 11.55%) and iris (62.7 +/- 5.94%) when compared with those of the normalizing reference gene beta2 microglobulin. The CB1 gene encodes a functional protein which is detected in its glycosylated (63 kDa) and unglycosylated (54 kDa) form in the same areas by a specific purified antibody raised against the amino terminus (residues 1-77) of the CB1 receptor. These results further support the proposed role of the CB1 receptor in controlling intraocular pressure, helping to explain the antiglaucoma properties of marijuana.

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.