HPV typing by in situ hybridization on cervical cytologic smears with ASCUS.

OBJECTIVE: To assess the prognostic significance of atypical squamous cells of undetermined significance (ASCUS) using an in situ hybridization (ISH) method for destined cervical cytologic smears and a cocktail of biotinylated DNA probes for human papillomavirus (HPV) 6, 11, 16, 18, 31 and 33. STUDY DESIGN: Two HPV DNA probe mixtures were applied to the same smear for the simultaneous detection of high-risk HPV types 16, 18, 31 and 33 and low-risk HPV 6 and 11. ISH was carried out on 192 smears. Among them, 59 showed koilocytosis, 91 ASCUS and 42 normal features. RESULTS: Low-risk HPV types were rarely found and associated mainly with koilocytosis (17%). High rates of potentially oncogenic HPV were detected in ASCUS (41%) and condyloma (73%). In addition, similar levels of positivity were found to be associated with ASCUS when using two probe mixtures specific to high-risk HPV: one included HPV genotypes 16 and 18 and the other, genotypes 31 and 33. CONCLUSION: HPV DNA typing by ISH on cervical cytologic smears might improve the identification of women at high risk of developing precancerous and cancerous cervical lesions.

Cyclic AMP as the second hormonal messenger in the in vitro folliculogenesis of the foetal thyroid gland of the rat.

Thyroid-stimulating hormone, the catecholamine isoproterenol, and prostaglandins E1 and E2, all substances known to increase cAMP concentration in thyroid tissue, accelerate the formation of follicular cavities in explanted thyroid of 15-day-old rat foetuses. Dibutyryl-cAMP added to the medium, but not sodium fluoride, also stimulates the folliculogenesis. Since fluoride stimulates membrane adenylate cyclase but does not increase the intracellular cAMP level, these results show that cAMP is involved as a second messenger in the activation of foetal thyroid morphogenesis induced by hormones. They indicate also that the thyroid gland of the foetal rat is capable of responding to hormonal stimulation as early as the 15th day of pregnancy; this implies that on day 15, the foetal thyroid possesses receptors not only for the thyroid-stimulating hormone, but also for catecholamines and prostaglandins.

Thyrotropin effects on vesicle transfer and thyroid follicle morphogenesis: a stereological study in the rat.

Incubation in a culture medium with and without TSH of 16 day-old foetal thyroid glands induces hypertrophy of the Golgi apparatus which may be correlated with a considerable increase in the number of secretory vesicles. A stereological study performed during the first 6 hr of incubation showed that: vesicle secretion was biphasic; vesicle secretion was heterogeneous with two different populations of vesicles; When TSH (20 mU and 80 mU) was added to the medium, the volume density of the follicular lumina increased; at least during the first 6 hr TSH seemed to be necessary to the formation of follicular lumina.

Basement membrane maintenance in cultures of isolated thyroid follicles.

Follicles from adult rat thyroid glands were isolated by digestion with collagenase followed by sieving. Electron microscopic studies have shown that basement membrane was present around each one of the isolated follicles. Follicles were cultured in vitro with or without thyrotropin and fibronectin. When cultured in a medium free of thyrotropin, basement membrane remained around them, while it was lacking in presence of this hormone.

Evidence for a role of the cytoskeleton in the in vitro folliculogenesis of the thyroid gland of the fetal rat.

Thyrotropic hormone (TSH) or cAMP accelerate the formation of follicular cavities in the explanted thyroid gland of the 15-day-old rat fetus. Cytochalasin B or vinblastine and nocodazole or colchicine, which disorganize microfilamental and microtubular structures respectively, inhibit or completely block in vitro-induced folliculogenesis. Exposure of the thyroid tissue to lumicolchicine, a structural isomer of colchicine deprived of antimicrotubular activity, does not inhibit the activation of folliculogenesis induced by TSH. These results are strong evidence for the supposition that microfilaments and microtubules are involved in the TSH-stimulated mechanisms resulting in thyroid folliculogenesis. Folliculogenesis requires the integrity of both microfilaments and microtubules.

Thyroid follicular morphogenesis mechanism: organ culture of the fetal gland as an experimental approach.

The morphological and physiological changes induced by organ culture and thyroid-stimulating hormone (TSH) stimulation in the rat fetal thyroid gland were studied. Organ culture increased Golgi activity which was further enhanced by TSH, subsequently facilitating the formation of intracellular lumina. TSH also raised the intracellular cAMP level. The intracellular lumina observed during follicular morphogenesis are structurally comparable to typical intracellular cavities formed in adult thyroid cells, which are considered as being the result of increased Golgi activity. The intracellular lumen, therefore, is probably not a physiologically significant step in thyroid morphogenesis.

Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells.

In situ hybridization was performed using cRNA probes on human liver biopsies to localize both positive and negative RNA strands of hepatitis C virus. From the 5' non-coding region of the viral genome, 210 bp, were amplified by reverse transcriptase-polymerase chain reaction and cloned in a plasmid. Probes were produced by in vitro transcription, and labeled using digoxigenin-11-UTP. Positive HCV-RNA strands were detected in all 20 of the patients analyzed, whereas negative strands were detected in only nine patients, as confirmed using computerized image analysis. Both probes labeled the cytoplasm of hepatocytes with a perinuclear intensification. Few of the mononuclear cells infiltrating the portal connective space contained positive HCV-RNA strands only. Stacks of dilated endoplasmic reticulum cisternae were observed by electron microscopy and their relationship with the infection was discussed. This study confirmed that non-radioactive in situ hybridization represents a useful tool to analyze the localization and replication of hepatitis C virus in liver tissue.