Role of the association of high-risk HPV identified by real-time PCR in cervical preneoplastic lesions.

OBJECTIVE: To evaluate the effects of infection in multiple types of high-risk human papilloma virus (HPV) in cervical preneoplastic lesions in patients undergoing colposcopy following a diagnosis of atypical squamous cells of unknown significance (ASCUS) and low-grade squamous intraepithelial (LSIL) cytology. MATERIALS AND METHODS: Between 2009 and 2010, 2,500 patients were recruited with a mean age of 35 +/- 5 years. Screening for cervical cancer was performed and in case of ASCUS and LSIL the patients underwent colposcopy. The tests for the detection and typing of viral DNA (HPV - DNA test) were performed on cervical swab with real-time PCR amplification. RESULTS: The prevalence of infection was 70% (1579/2256) in the patients recruited. In relation to the degree of preneoplastic lesions some high-risk HPV viral genotypes were identified: HPV 16 (319/1466), HPV 18 (164/1466), HPV 45 (76/1466), HPV 31 (215/1466), HPV 52 (145/1466), HPV 58 (55/1466) HPV 56 (79/1466), HPV 51 (110/1466), HPV 6(138/1466), HPV 11 (88/1466), HPV 42 (34/1466), HPV 53 (43/1466). In case of high-grade lesions of CIN (CIN2 and CIN3) a greater HPV co-infection was detected and in particular the association from 16 to 18 (70%), 16-33 (18%) and 16 to 52 (12%). CONCLUSIONS: Infection caused by the simultaneous presence of multiple HPV genotypes appears to be associated with a significantly increased risk of high-grade lesions of CIN or invasive cancer than the presence of single viral infections. The infection with multiple HPV types is a significant risk factor for high-grade lesions of CIN in women undergoing colposcopy for ASCUS cytology/LSIL. The use of real-time PCR has shown the ability not only to identify the different types of HPV, but also to monitor quantitatively the same over time, and during the study phase, to evaluate the sensitivity and specificity of the method in comparison with other techniques.

Mutations in TAZ/WWTR1, a co-activator of NKX2.1 and PAX8 are not a frequent cause of thyroid dysgenesis.

AIM: In 80-85% of cases, congenital hypothyroidism is associated with thyroid dysgenesis (TD), but only in a small percentage of cases mutations in thyroid transcription factors (NKX2.1, PAX8, FOXE1, and NKX2.5) have been associated with the disease. Several studies demonstrated that the activity of the transcription factors can be modulated by the interaction with other proteins, such as coactivators and co-repressors, and TAZ (transcriptional co-activator with PDZ-binding motif or WWTR1) is a co-activator interacting with both NKX2.1 and PAX8. In the present study we investigate the role of TAZ in the pathogenesis of TD. MATERIAL AND METHODS: By Single Stranded Conformational Polymorphism, we screened the entire TAZ coding sequence for mutations in 96 patients with TD and in 96 normal controls. RESULTS: No mutations were found in patients and controls, but we found several polymorphisms in both groups. No significant differences could be demonstrated in the prevalence of the mutations between patients and controls. CONCLUSIONS: Our data indicate that TAZ mutations are not a cause of TD in the series of patients studied.

Malignant melanoma metastasizing to the uterus in a patient with atypical postmenopause metrorrhagia. A case report.

UNLABELLED: The uterine metastases of melanoma are very rare. At the present time, only one case occurred in our department. CASE REPORT: a 54-year-old plurigravid woman showed a metrorrhagia of unknown origin. The patient underwent a diagnostic hysteroscopy and an endometrial biopsy, in order to investigate the symptomatic postmenopausal bleeding and exclude a neoplasia, such as the endometrial carcinoma. The patient was discharged with a diagnosis of uterine fibromatosis and called back to go through a complete laparoscopic hysterectomy and bilateral adnexectomy. During the operation, some metastases were found in the genital tract. An accurate physical examination allowed us to discover a cutaneous nevus, the excision and histology of which revealed its malignancy. The immunohistochemistry of the surgical sample was able to confirm the hypothesized relationship between the nevus and the metastases, thus leading to the diagnosis of malignant melanoma metastases, genital tract. It is important to make an accurate diagnostic passage to exclude tumoral pathology in patients with atypical uterine bleeding. Every uterine bleeding of the postmenopausal period (abnormal uterine bleeding, AUB) is considered atypical and it has to be early investigated, in order to exclude any endometrial cancer. The nature of the uterine bleedings can be ascribed to atrophy, dysfunctional matters (dysfunctional uterine bleeding, DBU), benign organic alterations, only in 7-10% of cases to endometrial cancer and more rarely to metastatic tumours, as well as this case of melanoma. Physicians should be aware of such unusual possibilities in order to look carefully for metastatic implants in adenomyomas.

Non-human primates: a model for tuberculosis research.

A variety of animals have been used for tuberculosis research, and each animal model has its strengths and weaknesses. We sought to develop a non-human primate model of tuberculosis to model aspects of human tuberculosis that are difficult to model in other animals, including the pathology in the lungs, various progression to disease, and immunologic correlates of infection or disease that are likely to be similar in humans. To date, we have infected 17 cynomolgus macaques (Macaca fasicularis) with a low dose (15-25CFU) of Mycobacterium tuberculosis strain Erdman. The monkeys were grouped into three categories on the basis of disease progression: rapid progression (advanced disease by 3 months post-infection), active/chronic infection (signs of disease but a slower progression), and latent infection (no signs of clinical disease). Animals were followed clinically post-infection, including blood work, physical examinations, serial bronchoalveolar lavage (BAL) and gastric aspirates for M. tuberculosis culture, chest radiographs, and tuberculin reactivity. Immunologic assays on cells from blood, BAL fluid, and tissue, have been performed, including proliferation, flow cytometry, ELIspot assays, cytotoxic T lymphocyte (CTL) assays, and ELISAs. The spectrum of disease observed in these monkeys is similar to humans, and this model may be very useful for studying pathogenesis and immunology of tuberculosis, as well as testing vaccines, diagnostic reagents, and drugs prior to use in human populations.

Herpesvirus-mediated systemic delivery of nerve growth factor.

Sustained systemic dissemination of therapeutic proteins from peripheral sites is an attractive prospect for gene therapy applications. Replication-defective genomic herpes simplex virus type 1 (HSV-1) vectors were evaluated for their ability to express nerve growth factor (NGF) as a model gene product both locally and systemically. Intra-articular inoculation of NGF expression vectors in rabbits resulted in significant increases in joint lavage and blood plasma NGF that persisted for 1 year. A rhesus macaque injected intra-articularly displayed a comparable increase in plasma NGF for at least 6 months, at which time the serum NGF levels of this animal were sufficient to cause differentiation of PC12 cells in culture, but not to increase footpad epidermis innervation. Long-term reporter transgene expression was observed primarily in ligaments, a finding confirmed by direct inoculation of patellar ligament. Patellar ligament inoculation with a NGF vector resulted in elevated levels of circulating NGF similar to those observed following intra-articular vector delivery. These results represent the first demonstration of sustained systemic release of a transgene product using HSV vectors, raising the prospect of new applications for HSV-1 vectors in the treatment of systemic disease.

Effect of mycobacterial infection on virus loads and disease progression in simian immunodeficiency virus-infected rhesus monkeys.

The effect of a mycobacterial infection on AIDS disease was studied in the simian model. Monkeys were infected with the primary virulent isolate SIV/DeltaB670 and inoculated 90 days later with BCG, an attenuated strain of Mycobacterium bovis. All monkeys experienced a dramatic transient increase in plasma viremia and CCR5 expression on T lymphocytes after BCG inoculation. Only two of the four SIV+ animals had substantial proliferative responses to PPD, with poor responders developing disseminated BCG during the course of the experiment. BCG inoculation of SIV-infected long-term nonprogressor (LTNP) monkeys was also performed. Similar to the acutely infected animals, two of three LTNPs experienced increases in plasma viral levels and CCR5 expression. In the majority of animals studied, there was no accelerated progression to AIDS despite the concomitant transient stimulation of virus replication and CCR5 expression on T lymphocytes.

Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines.

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.

Cardiovascular, respiratory, thermoregulatory, sedative, and analgesic effects of intravenous administration of medetomidine in rhesus macaques (Macaca mulatta).

BACKGROUND AND PURPOSE: Medetomidine is a selective, specific, and potent alpha2-adrenergic receptor agonist that has been utilized successfully as a sedative/analgesic agent in a variety of domestic and nondomestic animals. The objective of this study was to document the physiological effects of the intravenous administration of medetomidine in rhesus macaques (Macaca mulatta). METHODS: Fifteen healthy rhesus macaques (Macaca mulatta), 5 to 15 years old and weighing 5.5 to 11.8 kg, were given four dosages of medetomidine (50, 100, 150, and 200 microg/kg of body weight) intravenously, and cardiovascular, respiratory, thermoregulatory, sedative, and analgesic effects were determined. RESULTS: All four doses of medetomidine induced a similar and significant decrease in mean arterial pressure, as well as a transient but significant increase in respiratory rate followed by a longer-lasting significant decrease. Bradycardia, hypotension, and loss of thermoregulatory ability accompanied by a biphasic respiratory response and inconsistent sedation, analgesia, and muscular relaxation were observed. Heart rate decrease was rapid for all doses, but was significantly lower and of shorter duration after administration of the 50 microg/kg dosage. CONCLUSION: The inconsistency of the anesthetic plane induced by intravenous administration of medetomidine precludes it from being used alone to sedate rhesus macaques.

The origin and function of cement gland secretion in Pomphorhynchus laevis (Acanthocephala).

Cement gland protein in male and inseminated female individuals of an acanthocephalan parasite of fish, Pomphorhynchus laevis (Müller, 1776), was localized by immunohistochemistry using an antibody specific for cement protein. Male P. laevis possess 3 pairs of round to oval cement glands ranging from 0.5 to 0.9 mm in length and from 0.3 to 0.7 mm in width. Each gland has an outer portion containing nuclear fragments and other cellular organelles surrounding a space for storage of gland products. Very little work has been carried out on the nature of the cement gland secretions. We have previously reported that the major component of cement is a protein with molecular weight of 23 kDa; in fresh glands it is white in colour. Immunohistochemical studies herein reported were carried out using a polyclonal antibody raised against purified P. laevis p23 cement protein (anti-p23PL). Localization of p23 cement protein at the light microscope level, by means of the anti-p23PL antibody, shows that p23 is present within the cytoplasmic layer of the gland as well as in the gland duct lumen. Interestingly, the p23 cement protein was also identifiable at the posterior ends of females retaining the cap. Positivity to anti-p23PL antibody was obtained not only in the external part of the copulatory cap, but also within the vaginal tract and at the base of the uterine duct. Thus, we report herein the first photographic evidence that the copulatory cap is not a simple gonopore lid but it is really an intravaginal plug.

Immunocytochemical studies of the 5-HT(1A) receptor in ventral medullaryneurons that project to the intermediolateral cell column and contain serotonin or tyrosine hydroxylase immunoreactivity.

Activation of serotonin-1A receptors (5-HT(1A)R) in the medulla oblongata lowers sympathetic nerve discharge and blood pressure. Binding sites for 5-HT(1A)R ligands are present in ventral medullary nuclei [e.g., rostral ventrolateral medulla (RVLM), raphe pallidus (RPa), and parapyramidal region (PPR)] that project to sympathetic preganglionic neurons in the intermediolateral cell column (IML). However, the projections and the neurochemical contents of the ventral medullary neurons that are likely to be involved in the hypotensive actions of 5-HT(1A) agonists are unclear. Using a sheep antibody to a fragment of the third intracellular loop of the 5-HT(1A)R, we localized 5-HT(1A)R immunoreactivity (ir) to IML-projecting neurons that were retrogradely labeled with rhodamine beads injected into the IML of adult male rats. The percentages of IML-projecting neurons containing 5-HT(1A)R-ir were 49% in RPa, 34% in PPR, and 44% in RVLM. Using multiple-immunofluorescence labeling, we also demonstrated 5-HT(1A)R-ir in serotonergic (5-HT) and in catecholaminergic (tyrosine hydroxylase; TH-ir) neurons of the ventral medulla. The percentages of 5-HT-ir neurons containing 5-HT(1A)R-ir were 28% in RPa, 18% in PPR, and 31% in raphe obscurus. In addition, 5-HT(1A)R-ir was present in 14% of TH-ir neurons of the RVLM. Moreover, some IML-projecting neurons in the PPR and RPa were doubly immunolabeled for 5-HT(1A)R-ir and 5-HT, and some IML-projecting neurons in the RVLM were doubly immunolabeled for 5-HT(1A)R-ir and TH-ir. These data provide anatomical evidence for the presence of 5-HT(1A)R on serotonergic and catecholaminergic bulbospinal neurons and for their potential role in directly modifying the activity of these ventral medullary neurons.

The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.

Cytoskeletal organization in clusters of isolated polarized skate hepatocytes: structural and functional evidence for microtubule-dependent transcytosis.

Isolated hepatocytes from the marine vertebrate Raja erinacea (the little skate) retain their structural and functional integrity as clusters of cells formed around a single tubular bile canaliculus, and therefore can be used as a model of polarized hepatocytes in situ. In this study we used confocal and conventional epifluorescence microscopy in conjunction with fluorescent markers and immunocytochemistry to examine the structure and function of the cytoskeleton in these cells. Actin filaments in the hepatocyte clusters were found cortically and also concentrated in a pericanalicular array, while microtubules appeared to radiate away from a concentration near the apical membrane of the biliary pole towards the basolateral sinusoidal surfaces. Treatment of clusters with the microtubule disrupting agent, nocodazole, resulted in the microtubules depolymerizing from the basolateral surfaces towards the apical surface, indicating that the microtubules were oriented with their plus ends at the basolateral surface and their minus ends at the apical surface. Nocodazole was also found to disrupt the ability of clusters to transcytose a fluorescent bile salt derivative into their canalicular lumens. We detected cytoplasmic dynein in skate hepatocyte homogenates by Western blotting using an anti-dynein intermediate chain antibody, and immunofluorescent staining of intact hepatocytes revealed a punctate vesicular pattern. The polarized arrangement of microtubules, the presence of cytoplasmic dynein, and the inhibition of bile salt secretion by nocodozole are consistent with the microtubule cytoskeleton playing a fundamental role in the mediation of transcytosis, endocytosis, and bile excretory function in these hepatocytes. These polarized isolated skate hepatocytes represent an excellent experimental model for the in vitro study of hepatic transport, and allow for important comparative studies aimed at elucidating the evolutionarily conserved nature of various hepatocyte structures amongst the vertebrates.

Pre-operative axillary lymphoscintigraphy in breast cancer: experience with sub-areolar injection of 99Tcm-nanocolloidal albumin.

Thirty-two patients affected by monolateral breast cancer and five patients with benign mammary pathology were submitted to pre-operative lymphoscintigraphy by sub-areolar injection of nanocolloidal albumin with the aim of evaluating any axillary lymphonodular involvement. The 32 patients, affected by cancer, subsequently underwent a surgical operation so as to permit a pathological-anatomical study of the axillary cavity. A comparison between the clinical and instrumental examinations brought to light the much greater reliability of the latter which gave 87.5% of correct diagnoses. The authors emphasize the absence of false positive results and the excellent sensitivity of the method in bringing to light even limited involvements of the axillary lymphonodes. The authors believe that axillary lymphoscintigraphy deserves to be included in the pre-operative protocols which afford a correct staging of the disease and, consequently, a better assessment of the extent of the surgical action.

[Sequential hepato-biliary scintigraphy and percutaneous cholangiography in the diagnosis of benign diseases of the biliary tract Indications and limits]. / La scintigrafia sequenziale epato-biliare e la colangiografia percutanea nella diagnosi delle affezioni benigne della via biliare. Indicazioni e limiti.

Attention is drawn to the results of a study of 45 patients with bile duct calculosis designed to assess the soundness and the limits of two diagnostic examinations (HIDA and PTC) in determining the morphological and functional characteristics of the hepatoenteric tract. The results indicated that HIDA is a useful first step in average to serious cases of icterus as a means of evaluating the functional state of liver cells and the bile ducts by determination of activity-time curves for preselected areas, whereas PTC is more important in cases of serious icterus, since it also enables the patient to be suitably prepared for surgery.