Heat SHOCK proteins in equine spermatozoa: Expression and correlation to kinetic and environmental parameters.

Heat Shock Proteins are chaperones primary involved in the repair of cellular damages induced by temperature. The harmful effect of temperature on the male gonad is well known, on the contrary knowledge on the effects of the environment on semen quality are still insufficient. The aim of this paper was to learn more about the role of HSPs and the environment in modulating the physiology of equine male gonads. We showed a detailed analysis of equine semen characteristic and the expression level of three HSPs (60-70-90) over a one-year period analyzing the effects of temperature and humidity and the correlation among the different variables. We showed also that the interpretation of results depends strongly on the way in which data are assembled and analyzed, therefore we compared results obtained from three different ways of grouping: according to single months, to weather seasons and to mare reproductive periods. Results showed that the expression of the three HSPs is correlated to the environment through temperature and humidity and that it reaches the highest level in the breeding season and in summer. We found also that HSPs expression is correlated to some variables describing the quality of equine semen (concentration) and the kinetic of spermatozoa (total motility-MOT, %, average path velocity -VAP, µm/s- and lateral head displacement -ALH, µm). No correlation was found between HSPs expression and the mitochondrial membrane potential; while viability and HSP90 expression resulted positively correlated. The month-by-month analysis evidenced that in February equine semen has the highest kinetic characteristics (increased linearity -LIN, %-, straightness -STR, % -and average path velocity -VAP, µm/s) with the highest number of motile, progressive motile and rapid cells. These results may have a great impact in the comprehension of functional aspects of the physiology of equine semen and may have potential implications for breeders who want to understand the period (and/or month) of the year in which equine semen reaches the best characteristics with increased chances for better results in reproductive practice.

Localization and functional modification of L-type voltage-gated calcium channels in equine spermatozoa from fresh and frozen semen.

It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level focusing on the function of L-type calcium channels. We report that in cryopreserved spermatozoa the mean basal value of [Ca(2+)]i is higher than that of spermatozoa from fresh semen (447.130 vs. 288.3 nM; P < 0.001) and L-type channels function differently in response to their agonist and antagonist in relation to semen condition (fresh or frozen-thawed). We found that on addition of agonist to the culture medium, the increase in intracellular calcium concentrations ([Ca(2+)]i) was greater in frozen semen than in fresh semen (Δ[Ca(2+)]i = 124.59 vs. 16.04 nM; P < 0.001), whereas after the addition of antagonist the decrease in [Ca(2+)]i was lower in frozen semen than in fresh semen (Δ[Ca(2+)]i = 32.5 vs. 82.5 nM; P < 0.001). In this article, we also discuss the impact of cryopreservation on sperm physiology.

Effects of the environment on dog semen parameters and testosterone concentration.

Whether a mammal reproduces seasonally or continuously depends mostly on the environment and its effects on the endocrine pattern. Although the dog was the first species to have been domesticated, little information is available on the changes in reproduction that have occurred since. In this study, we evaluated whether environmental stimuli can act as modulators of male gonadal activity in the dog at the latitude of Bari (Italy). Therefore, for 1 year, serum and seminal testosterone (T) concentrations, together with seminal parameters, were recorded monthly and evaluated in relation to environmental variables such as temperature, humidity, and photoperiod. We found that, in temperate regions, the annual serum T profile is not affected by environmental conditions, whereas seminal T profile peaks in October and reaches its nadir in April. The percentage of progressive motile spermatozoa is also dependent on environmental cues. The results support the intuitive idea that recorded data require a proper analysis to be meaningful. In fact, we found that, in the dog, environmental changes appear to affect male gonadal physiology, and this is clear when recorded data are analyzed monthly; in contrast, pooling data into seasonal groups hides monthly environmental variations.

The expression of leptin receptor in the ovary of the queen: leptin receptor expression in queen ovary.

Leptin is a Ob gene product secreted mainly by adipose tissue. Several reports showed leptin production by other tissue including the ovary. The action of leptin is mediated upon binding to its receptor widely expressed in reproductive tissues in different species. In fact, there are growing evidences that leptin plays an important role in the modulation of reproductive functions. Therefore, the aim of this study was to evaluate in the queen, the expression of leptin receptor during the functional ovarian cycle and pregnancy. We found that the ovaries of the queen express leptin receptor in all the examined phases. The highest leptin receptor expression was found in the luteal phase (pseudopregnancy, pregnancy) compared to other phases of the cycle (anestrus, proestrus, estrus). The variations in the expression of leptin receptor suggest a likely implication of leptin in the modulation of ovarian activity, in the examined species.

Leptin and queen ovary: new insights about ovulation.

Leptin has been proven to be crucial in the ovulatory process. Aims of this study are to assess the expression of leptin receptor (Ob-R) in the ovaries of queens at estrus and to evaluate the capability of leptin in modulating ovarian contractility in vitro. Right ovaries underwent immunoblot analysis. Left ovaries were mounted in an organ bath under physiological condition and exposed to murine leptin (10(-6) M). Immunoblot analysis showed that the queen ovary expresses leptin receptor at estrus. Leptin at the dose of 10(-6) M significantly reduced the contractile activity of the ovary. The presence of ovarian Ob-R and leptin inhibitory effects on ovarian contractility suggest leptin implication in the modulation of ovarian activity, as well as in ovulatory disorders.

Changes in the expression of the miu-opioid receptor in the mare oviduct during oestrus and anoestrus.

The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed MOR in the apical cytoplasm and, except for the isthmus of oestrous mares, also in the nucleus. Non-ciliated cells showed MOR only in the isthmus segment during oestrus. Stromal cells showed different immunoreactivity along the oviduct segments and during the oestrous and anoestrous phases. The myosalpinx displayed immunostained myocytes in the intrinsic musculature of the ampulla and in the extrinsic and intrinsic musculature of the isthmus without significant differences between anoestrus and oestrus. Blood vessels expressed MOR in endothelial cells and smooth muscle cells in the isthmus myosalpinx of oestrous mares only. In conclusion, these findings show diverse MOR expression in the three segments constituting the oviduct, as well as changes in MOR expression linked to the mare's physiological condition.

Expression of the micro-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production.

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions.

Expression of the mu opioid receptor and effects of the opioid antagonist naloxone on in vitro maturation of oocytes recovered from anoestrous bitches.

The mu-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus-oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 x 10(-6), 1 x 10(-8) and 1 x 10(-10) M. The rate of oocytes resuming meiosis after culture in presence of 1 x 10(-6) M Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as delta and kappa, and possible interactive effects of their antagonists on canine oocyte maturation.

The presence of the mu-opioid receptor in the isthmus of mare oviduct.

The presence of the mu-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (N-glycosidase F and O-glycosidase) and chemical (beta-elimination) treatments. Immunoblotting analysis showed that the mu-opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by O-glycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the mu-opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galbeta(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the mu-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the mu-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after N-glycosidase F treatment, whereas it was preserved in the apical region after beta-elimination. Most non-ciliated cells expressed the mu-opioid receptor with both N- and O-linked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and beta-elimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the mu-opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the mu-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the mu-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective condition of the sperm in the mare oviduct.

Relationship of body mass index to early complications in knee replacement surgery.

BACKGROUND: The purpose of this study was to evaluate the relationship between body mass index and early complications following knee replacement. METHODS: Five hundred and twenty-seven patients who underwent primary knee replacement were recruited. All these patients were subjected to a pre-operative assessment and follow-up at 6 weeks and 1 year following surgery. Any complication occurring during this period was recorded. Complications were grouped into systemic and local, minor and major. RESULTS: 12.1% (N = 64) patients had an early complication following knee replacement surgery; 9.2% (N = 48) patients had a major complication. Overall BMI did not seem to influence significantly the rate of complication with a P-value of 0.338. A stronger correlation was found between the surgeon and presence of complication with a P-value of 0.004. CONCLUSIONS: BMI has a weak correlation to early complications following joint replacement surgery. The operating surgeon seems to have a stronger correlation to early complications as compared with BMI.

Rate of computed tomography pulmonary angiographies (CTPA) positive for pulmonary embolism and predictive scores.

PURPOSE: In March 2005, the Simplified Wells Scoring System was introduced as a predictive tool for pulmonary embolism at Hairmyres Hospital in Scotland. This study was conducted to evaluate the efficacy of the Wells score in predicting the presence of pulmonary embolism in terms of rate of positive computed tomography pulmonary angiographies (CTPAs). MATERIALS AND METHODS: Two hundred patients with suspected pulmonary embolism were retrospectively reviewed using a sequential method: 100 patients before implementation of the Wells score and a further 100 after its introduction. RESULTS: The rate of positive CTPAs for pulmonary embolism changed from 23% before to 27% after the introduction of the Wells Scoring System. CONCLUSIONS: The rate of positive CTPAs did not change significantly after the introduction of the Wells score.

Relationship of body mass index to early complications in hip replacement surgery : study performed at Hinchingbrooke Hospital, Orthopaedic Directorate, Huntingdon, Cambridgeshire.

The purpose of this study was to evaluate the relationship between body mass index and early complications following total hip replacements. Five hundred and fifty patients who underwent primary total hip replacement were recruited. All these patients were subjected to a pre-operative assessment and follow-up at 6 weeks and 1 year following surgery. Any complications occurring during this period were recorded. Complications were grouped into systemic and local, both subdivided into minor and major depending on the risk involved. Fifty-six patients (10.2%) had an early complication following hip replacement surgery. Forty-four patients (8%) had a major local complication. Overall, there did seem to be a weak correlation between BMI and the rate of complications, with a p value of 0.104. A correlation was also found between the surgeon and presence of complications with a p value of 0.736. There is a weak correlation between BMI and early complications following hip replacement surgery, and there also seems to be a correlation between the operating surgeon and early complications, but this is not statistically significant.

Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro.

Opioid peptides are the most effective drugs in controlling pain; their action is elicited by binding to specific membrane receptors. The gastrointestinal tract represents, after the nervous system, the site in which the opioid receptors are expressed at high levels. The opioid agonist morphine has a significant inhibitory effect on intestinal motility, this action is blocked by naloxone an opioid antagonist mainly active at mu and kappa receptors. In this study the presence of mu opioid receptor on rabbit jejunum was investigated by western blot. The effects of beta-endorphin, the endogenous opioid peptide with the highest affinity to the mu opioid receptor and those of naloxone on spontaneous rabbit jejunum contractions were evaluated. Beta-endorphin (10(-6) M) showed a relaxant effect on jejunum contractility while naloxone showed a dual effect inducing an increase of spontaneous contractility at low concentrations (10(-6) M, 10(-7) M, 10(-8) M) and a decrease when high concentrations (10(-3) M, 10(-4) M, 10(-5) M) were utilized. The obtained results demonstrate that mu opioid receptor is expressed in rabbit jejunum and suggest that this receptor may be involved in mediating the effects of both opioid agonist and antagonist on jejunum contractions.

Effects of beta-endorphin and Naloxone on in vitro maturation of bovine oocytes.

Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.

Naloxone inhibits A6 cell Na(+)/H(+) exchange by activating protein kinase C via the mobilization of intracellular calcium.

The opioid receptor antagonist, naloxone, has been shown to have beneficial effects in the kidney and to be implicated in renal salt and water balance. In the present study the signal transduction pathways utilized by naloxone were studied in an epithelial cell line model of the cortical collecting duct, A6 cells. We found that naloxone has a dual effect depending on the concentration used: at a low concentration (10(-6) M) it antagonized the beta-endorphin-dependent increase in cytoplasmic calcium [Ca(2+)](i), while at higher concentrations (>10(-5) M) it increased [Ca(2+)](i) and intracellular inositol phosphate levels. While naloxone-induced increases in [Ca(2+)](i) occurred in the absence of external calcium, it was significantly stimulated by increasing the external calcium concentration, suggesting that naloxone increases [Ca(2+)](i) via both calcium release and calcium influx. In polarized A6 cell monolayers naloxone inhibited the activity of the Na(+)/H(+) exchanger (NHE) only when added to the basolateral cell surface. This inhibition of the NHE was prevented by pretreatment of the cells with either the intracellular calcium chelator, BAPTA or with the protein kinase C inhibitor, calphostin C. These findings demonstrate that naloxone induces a rapid increase in intracellular calcium which inhibits the NHE via the calcium-dependent protein kinase C regulatory pathway.

Immunohistochemical reactivity of anti-melanoma monoclonal antibody 225.28S in human breast cancer biopsies.

The high molecular weight melanoma-associated antigen, defined by murine monoclonal antibody (IgG1) 225.28S is largely expressed by melanoma cells and weakly expressed by other human tumors originating from neural crest. In this study, we analyzed the immunohistochemical reactivity of MoAb 225.28S in human breast cancer biopsies. A total of 92 breast cancer biopsies (66 infiltrating lobular and 26 infiltrating ductal carcinomas) were initially tested along with 26 melanomas (positive controls), 23 gastric/colonic adenocarcinomas and 13 neuroendocrine tumors. Forty-four out of 66 lobular breast carcinomas showed positive immunostaining with 225.28S MoAb as well as only 6 out of 26 infiltrating ductal histotype and 12 out of 26 melanomas. Conversely, gastric and colonic adenocarcinomas and neuroendocrine tumors were completely negative. The pattern of positivity in breast carcinomas was associated with malignant cells, rather than with the stroma or histiocytes infiltrating the lesions. Nonspecific cross-reactivity of 225.28S with breast carcinomas was excluded using a similar murine antithyreoglobulin MoAb, which gave negative staining in all biopsies. These results indicated that HMW-MAA or a similar sequence recognized by 225.28S MoAb is often expressed by lobular breast carcinomas but rarely by ductal adenocarcinomas. This seems to suggest that lobular breast carcinoma has common "ancestor" antigens with melanoma.

Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region.

Tubular adenoma of the breast: an immunohistochemical study of ten cases.

Ten cases of so-called "tubular" adenoma and six cases of fibroadenoma of the breast have been investigated with an immunohistochemical technique with the aim of providing both more details on their immunophenotype and of ascertaining the possible relationships between tubular adenoma and fibroadenoma. Smooth Muscle Actin, Cytokeratin 14, GFAP, S-100 Protein and Vimentin immunoreactivity have clearly demonstrated that cells with myoepithelial immunophenotype are one of the major cell components in breast adenomas. Epithelial Membrane Antigen (EMA), Human Milk Fat Globule II (HMFG II), Estrogen and Progesterone receptors have been detected in adluminal epithelial cells exclusively. Furthermore, Smooth Muscle Actin and Vimentin highlighted an abundant myofibroblastic component, intermingled with tubular structures in both tumor types. A low percentage (10-22%) of adluminal cells and of myofibroblasts showed Ki-67 immunoreactivity in tubular adenomas and in fibroadenomas, whereas only rare myoepithelial cells demonstrated Ki-67 positivity in both tumor types. These data seem to indicate that several cell components of both epithelial and mesenchymal origin (epithelial cells, myoepithelial cells, myofibroblasts) are involved in the genesis of tubular adenomas. The morphological and immunohistochemical features of tubular adenomas closely resemble, in some areas of the tumors, those of fibroadenoma. Therefore, they may represent histogenetically related neoplasms with exuberant ductular component in tubular adenomas and predominant stromal component in fibroadenoma.

Polymerase chain reaction in liposomes.

BACKGROUND: Compartmentalization of biochemical reactions within a spherically closed bilayer is an important step in the molecular evolution of cells. Liposomes are the most suitable structures to model this kind of chemistry. We have used the polymerase chain reaction (PCR) to demonstrate that complex biochemical reactions such as DNA replication can be carried out inside these compartments. RESULTS: We describe the first example of DNA amplification by the PCR occurring inside liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or of a mixture of POPC and phosphatidylserine. We show that these liposomes are stable even under the high temperature conditions used for PCR. Although only a very small fraction of liposomes contains all eight different reagents together, a significant amount of DNA is produced which can be observed by polyacrylamide gel electrophoresis. CONCLUSIONS: This work shows that it is possible to carry out complex biochemical reactions within liposomes, which may be germane to the question of the origin of living cells. We have established the parameters and conditions that are critical for carrying out this complex reaction within the liposome compartment.

[Adenolymphoma of the salivary glands. The anatomicoclinical, epidemiological and therapeutic aspects of 76 cases]. / Adenolinfoma della ghiandole salivari. Aspetti anatomo-clinici, epidemiologici e terapeutici di 76 casi.

A series of 76 cases of adenolymphoma (Warthin's tumor) of the salivary glands is presented with emphasis on its epidemiological, clinico-pathological and therapeutic features; together with the analysis of the clinical records and of the epidemiological data, this study has reevaluated the histopathological features of the 76 cases of Warthin's tumor on hematoxylin-eosin stained slides. On these bases, the authors underline the frequent multifocality and/or bilaterality of Warthin's tumor (11.3% of cases), its frequent association with tumors of other sites (11.3% of cases), the prevalence in males (M:F ratio = 24:1) and in adults (mean age: 57 years) of this type of tumor. Furthermore, this study confirms the parotid gland as the elective site for Warthin's tumor and the possibility to categorize this entity into four different histological types. The latter finding should be kept in mind especially when fine needle aspiration (FNA) is used as a preoperative diagnostic procedure for Warthin's tumor to avoid misleading diagnosis and overtreatment. From the analysis of the results the authors underline the need for a complex and multidisciplinary approach to the diagnosis of Warthin's tumor in order to allow conservative surgery with preservation of the facial nerve.